Dihydroxybenzamide compound having HSP90 inhibitory activity or pharmaceutically acceptable salt thereof, and medical use thereof

ABSTRACT

Disclosed is a novel compound having HSP90 inhibitory activity or a pharmaceutically acceptable salt thereof, and a medicinal use thereof, and composition comprising a dihydroxyphenyl compound or a benzamide compound, which is a novel compound having the HSP90 inhibitory activity of the present invention can effectively inhibit HSP90, and thus can be usefully used as a pharmaceutical composition for preventing or treating HSP90-mediated diseases or a health functional food for preventing or improving HSP90-mediated diseases, which selected from the group consisting of cancer diseases, degenerative neurological diseases and viral infections.

CROSS REFERENCE TO PRIOR APPLICATIONS

This application is a Continuation Application of U.S. patentapplication Ser. No. 16/073,342 filed on Jul. 27, 2018 under 35 U.S.C. §120, which is the 35 U.S.C. 371 national stage of Internationalapplication PCT/KR2017/001034 filed on Jan. 31, 2017, which claimspriority to Korean applications 10-2016-0011626 filed on Jan. 29, 2016and 10-2016-0011622 filed on Jan. 29, 2016, the entire contents of eachof the above-identified applications are hereby incorporated byreference.

BACKGROUND

The present invention relates to a novel compound having HSP90inhibitory activity or a pharmaceutically acceptable salt thereof and amedical use thereof.

A HSP90 protein is one of the most abundant chaperones within eukaryoticcells and is responsible for stabilization and activity regulation ofvarious proteins related to cell growth differentiation and survival.The substrate protein of the HSP90 which is called the client proteincontains over 50 cancer-inducing proteins. If the HSP90 activity isinhibited, the HSP90 client proteins are degraded by the proteasome.

Therefore, the HSP90 activity inhibitor can decrease the activity ofvarious cancer-inducing proteins at the same time and thus it hasattracted great attention as an anticancer agent capable of beingapplied to a wide variety of cancers. In particular, HSP90 has beenreported to be effective treatment of cancer with resistance, because itsimultaneously reduces activity of various cancer-inducing proteins.

In addition, it has been reported that HSP90 inhibitor may be used as atherapeutic agent for degenerative neurological diseases, becauseproteins that cause degenerative nerve diseases are also present in theHSP90 client proteins.

The HSP90 inhibitor started with the development of the naturalsubstance geldanamycin (GA). GA has been found to lead to thedecomposition of Src, the client protein via inhibition of HSP90 in 1994and thereafter inhibitors of targeting HSP90 have been developedactively. However, GA has a strong anticancer effect, but has problemsof liver toxicity, solubility and stability. To compensate it. GAderivatives such as Tanespimycin (17-AAG), alvespimycin (17-DMAG) andretaspimycin are developed, but the problem has not been solved by thestructural characteristics of GA. HSP90 inhibitors of various structureshave been researched at the clinical stage, but since FDA-approved drughas not yet been developed, new and strong efficacious compounds arerequired.

Accordingly, an object of the present invention is to provide a noveldihydroxyphenyl compound or a benzamide compound.

Also, another object of the present invention is to provide apharmaceutical composition for preventing or treating HSP90-mediateddiseases comprising a novel dihydroxyphenyl compound or benzamidecompound, as an active ingredient.

In addition, another object of the present invention is to provide ahealth functional food for preventing or improving HSP90-mediateddisease comprising a novel dihydroxyphenyl compound or benzamidecompound, as an active ingredient.

SUMMARY

In order to achieve the above object, the present invention provides adihydroxyphenyl-based compound represented by following Chemical Formula1, a stereoisomer thereof, a racemic mixture thereof or apharmaceutically acceptable salt thereof:

in Chemical Formula 1,

R₁ is any one selected from the group consisting of halogen, C1-C4 alkyland C1-C4 alkoxy,

R₂ is any one selected from the group consisting of C3-C6 cycloalkyl,phenyl, halogen, C1-C4 alkyl and C1-C4 alkoxy, and

R₃ is C1-C4 alkyl or C1-C4 alkoxy.

Also, the present invention provides a pharmaceutical composition forpreventing or treating heat shock protein 90 (HSP90)-mediated diseasecomprising a dihydroxyphenyl-based compound represented by the aboveChemical Formula 1, a stereoisomer thereof, a racemic mixture thereof ora pharmaceutically acceptable salt thereof, as an active ingredient.

In addition, the present invention provides a health functional food forpreventing or improving heat shock protein 90 (ISP90)-mediated diseasecomprising a dihydroxyphenyl-based compound represented by the aboveChemical Formula 1, a stereoisomer thereof, a racemic mixture thereof ora pharmaceutically acceptable salt thereof, as an active ingredient.

Further, the present invention provides a benzamide compound representedby following Chemical Formula 2 or a pharmaceutically acceptable saltthereof:

In Chemical Formula 2,

R₁ is halogen or C1-C4 alkyl,

R₂ is

each of R₃ and R₄ can be the same as or different from to each other andis any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy,

each of R₅ and R₆ can be the same as or different from to each other andis any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy, or R₅ and R₆ are connected to each other to form 5- or6-membered ring,

R₇ is any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy,

R₈ is H or halogen,

R9 is any one selected from the group consisting of C1-C4 alkyl, C1-C4alkoxy and benzyl groups.

Also, the present invention provides a pharmaceutical composition forpreventing or treating heat shock protein 90 (HSP90)-mediated diseasecomprising a benzamide compound represented by the above ChemicalFormula 2 or a pharmaceutically acceptable salt thereof, as an activeingredient.

In addition, the present invention provides a health functional food forpreventing or improving heat shock protein 90 (HSP90)-mediated diseasecomprising a benzamide compound represented by the above ChemicalFormula 2 or a pharmaceutically acceptable salt thereof, as an activeingredient.

A composition comprising a dihydroxyphenyl compound or a benzamidecompound, which is a novel compound having the HSP90 inhibitory activityaccording to the present invention, can effectively inhibit HSP90, andthus is useful as a pharmaceutical composition for preventing ortreating HSP90-mediated disease or a health functional food forpreventing or improving HSP90-mediated disease, which is selected fromthe group consisting of a cancer disease, a degenerative neurologicaldisease and a viral infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of confirming HSP90 inhibitory activity byCompound 17 according to Example 1 of the present invention.

FIG. 2 illustrates (A) showing the results of inhibiting theproliferation rate of non-small cell lung cancer cells by variousconcentrations of Compound 17, and (B) showing a result of confirmingthe inhibition of survival of non-small cell lung cancer cells.

FIG. 3 shows the results of inhibition of colony formation of non-smallcell lung cancer cells of Compound 17 at various concentrations.

FIG. 4 shows the results of changes in protein expression after treatingCompound 17 having various concentrations on non-small cell lung cancercells.

FIG. 5 is a graph showing the HSP90 inhibitory effect of the2,4-dihydroxy-5-isopropy-methyl-N-(4-(propylcarbamoyl)benzyl) benzamideof compounds according to the present invention.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail.

The inventors of the present invention have studied a compound showingheat shock protein 90 (HSP90) inhibitory effect and synthesized acompound represented by the following formula 1 and completed thepresent invention by confirming its HSP90 inhibitory effect.

Accordingly, the present invention provides a dihydroxyphenyl-basedcompound represented by following Chemical Formula 1, a stereoisomerthereof, a racemic mixture thereof or a pharmaceutically acceptable saltthereof:

wherein R₁ is any one selected from the group consisting of halogen,C1-C4 alkyl and C1-C4 alkoxy,

R₂ is any one selected from the group consisting of C3-C6 cycloalkyl,phenyl, halogen, C1-C4 alkyl and C1-C4 alkoxy, and

R₃ is C1-C4 alkyl or C1-C4 alkoxy.

In the dihydroxyphenyl-based compound represented by the ChemicalFormula 1, R₁ is C1-C4 alkyl, R₂ is phenyl and R₃ is C1-C4 alkyl.

The present invention also relates to a pharmaceutical composition forpreventing or treating heat shock protein 90 (HSP90)-mediated diseasecomprising a dihydroxyphenyl-based compound represented by followingChemical Formula 1, a stereoisomer thereof, a racemic mixture thereof ora pharmaceutically acceptable salt thereof, as an active ingredient:

wherein R₁ is any one selected from the group consisting of halogen,C1-C4 alkyl and C1-C4 alkoxy,

R₂ is any one selected from the group consisting of C3-C6 cycloalkyl,phenyl, halogen, C1-C4 alkyl and C1-C4 alkoxy, and

R₃ is C1-C4 alkyl or C1-C4 alkoxy.

In the dihydroxyphenyl-based compound represented by the ChemicalFormula 1, R₁ is C1-C4 alkyl, R₂ is phenyl and R₃ is C1-C4 alkyl.

The heat shock protein 90-mediated disease is one or more diseasesselected from the group consisting of cancer diseases, degenerativeneurological diseases and viral infections.

The cancer can be selected from the group consisting of non-small celllung cancer, breast cancer, ovarian cancer, uterine cancer, pancreaticcancer, lung cancer, gastric cancer, liver cancer, colon cancer, skincancer, head or neck cancer, brain cancer, larynaeal cancer, prostatecancer, bladder cancer, esophageal cancer, thyroid cancer, kidneycancer, rectal cancer, acute alyelogenous leukemia, chronic myelogenousleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia andblood cancer, but is not limited thereto.

The degenerative neurological disease is selected from the groupconsisting of stroke, paralysis, memory loss, memory impairment,dementia, forgetfulness, Parkinson's disease, Alzheimer's disease,Pick's disease, Creutzfeld-Kacob disease, Huntington's disease andamyotrophic lateral sclerosis, but is not limited thereto.

Also, the present invention also relates to a health functional food forpreventing or improving heat shock protein 90 (HSP90)-mediated diseasecomprising a dihydroxyphenyl-based compound represented by followingChemical Formula 1, a stereoisomer thereof, a racemic mixture thereof ora pharmaceutically acceptable salt thereof, as an active ingredient:

wherein R₁ is any one selected from the group consisting of halogen,C1-C4 alkyl and C1-C4 alkoxy,

R₂ is any one selected from the group consisting of C3-C6 cycloalkyl,phenyl, halogen, C1-C4 alkyl and C1-C4 alkoxy, and

R₃ is C1-C4 alkyl or C1-C4 alkoxy.

Also, the inventors of the present invention also studied a compoundshowing Heat Shock Protein 90 (HSP90) inhibitory effect, and synthesizeda compound represented by the following Chemical Formula 2 and confirmedits HSP90 inhibitory effect to complete the present invention.

Accordingly, the present invention provides a benzamide compoundrepresented by following Chemical Formula 2 or a pharmaceuticallyacceptable salt thereof:

wherein R₁ is halogen or C1-C4 alkyl,

R₂ is

each of R₃ and R₄ can be the same as or different from to each other andis any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy,

each of R₅ and R₆ can be the same as or different from to each other andis any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy, or R₅ and R₆ are connected to each other to form 5- or6-membered ring.

R₇ is any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy.

R₈ is H or halogen,

R₉ is any one selected from the group consisting of C1-C4 alkyl, C1-C4alkoxy and benzyl groups.

In the benzamide compound represented by the Chemical Formula 2, R₁ ishalogen or C1-C4 alkyl, and R₂ is

R₃ is any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy, and R₉ is any one selected from the group consisting ofC1-C4 alkyl, C1-C4 alkoxy and benzyl groups.

The benzamide compound represented by the Chemical Formula 2 can be anyone selected from the group consisting ofN-benzyl-5-chloro-2,4-dihydroxybenzamide,N-benzyl-5-chloro-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-(4-methoxy benzyl)-N-methylbenzamide,5-chloro-N-(3,4-dimethoxybenzyl)-2,4-dihydroxy-N-methylbenzamide,N-(benzo[d][1,3]dioxol-5-ylmethyl)-5-chloro-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(3,4,5-trimethoxybenzyl)benzamide,5-chloro-N-(2-chloro-3,4,5-trimethoxybenzyl)-2,4-dihydroxy-N-methylbenzamide,N-(2-bromo-3,4,5-trimethoxybenzyl)-5-chloro-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(3-methylbenzyl)benzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(2-methylbenzyl)benzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(4-methylbenzyl)benzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(4-(methylcarbamoyl)benzyl)benzamide,5-chloro-N-(4-(ethylcarbamoyl)benzyl)-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(4-(propylcarbamoyl)benzyl)benzamide,2,4-dihydroxy-5-isopropyl-N-methyl-N-(4-(methylcarbamoyl)benzyl)benzamide,N-(4-(ethylcarbamoyl)benzyl)-2,4-dihydroxy-5-isopropyl-N-methylbenzamide,2,4-dihydroxy-5-isopropyl-N-methyl-N-(4-(propylcarbamoyl)benzyl)benzamideand N-benzyl-2,4-dihydroxy-5-isopropyl-N-methylbenzamide and preferably2,4-dihydroxy-5-isopropyl-N-methyl-N-(4-(propylcarbamoyl)benzyl)benzamide.

In addition, the present invention also provides a pharmaceuticalcomposition for preventing or treating heat shock protein 90(HSP90)-mediated disease comprising a benzamide compound represented byfollowing Chemical Formula 2 or a pharmaceutically acceptable saltthereof, as an active ingredient:

wherein R₁ is halogen or C1-C4 alkyl,

R₂ is

each of R₃ and R₄ can be the same as or different from to each other andis any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy,

each of R₅ and R₆ can be the same as or different from to each other andis any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy, or R₅ and R₆ are connected to each other to form 5- or6-membered ring,

R₇ is any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy,

R₈ is H or halogen,

R₉ is any one selected from the group consisting of C1-C4 alkyl, C1-C4alkoxy and benzyl groups.

In the benzamide compound represented by the Chemical Formula 2, R₁ canbe halogen or C1-C4 alkyl, and R₂ is

R₃ can be any one selected from the group consisting of H, C1-C4 alkyland C1-C4 alkoxy, and R₉ can be any one selected from the groupconsisting of C1-C4 alkyl, C1-C4 alkoxy and benzyl groups.

The benzamide compound represented by the Chemical Formula 2 can be anyone selected from the group consisting ofN-benzyl-5-chloro-2,4-dihydroxybenzamide,N-benzyl-5-chloro-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-(4-methoxybenzyl)-N-methylbenzamide,5-chloro-N-(3,4-dimethoxybenzyl)-2,4-dihydroxy-N-methylbenzamide,N-(benzo[d][1,3]dioxol-5-ylmethyl)-5-chloro-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(3,4,5-trimethoxybenzyl)benzamide,5-chloro-N-(2-chloro-3,4,5-trimethoxybenzyl)-2,4-dihydroxy-N-methylbenzamide,N-(2-bromo-3,4,5-trimethoxybenzyl)-5-chloro-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(3-methylbenzyl)benzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(2-methylbenzyl)benzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(4-methylbenzyl)benzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(4-(methylcarbamoyl)benzyl)benzamide,5-chloro-N-(4-(ethylcarbamoyl)benzyl)-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(4-(propylcarbamoyl)benzyl)benzamide,2,4-dihydroxy-5-isopropyl-N-methyl-N-(4-(methylcarbamoyl)benzyl)benzamide,N-(4-(ethylcarbamoyl)benzyl)-2,4-dihydroxy-5-isopropyl-N-methylbenzamide,2,4-dihydroxy-5-isopropyl-N-methyl-N-(4-(propylcarbamoyl)benzyl)benzamideand N-benzyl-2,4-dihydroxy-5-isopropyl-N-methylbenzamide and preferably2,4-dihydroxy-5-isopropyl-N-methyl-N-(4-(propylcarbamoyl)benzyl)benzamide.

The heat shock protein 90 mediated disease is at least one selected fromthe group consisting of cancer diseases, degenerative neurologicaldiseases and viral infections.

The cancer disease can be any one selected from the group consisting ofnon-small cell lung cancer, breast cancer, ovarian cancer, uterinecancer, pancreatic cancer, lung cancer, gastric cancer, liver cancer,colon cancer, skin cancer, head or neck cancer, brain cancer, laryngealcancer, prostate cancer, bladder cancer, esophageal cancer, thyroidcancer, kidney cancer, rectal cancer, acute myelogenous leukemia,chronic myelogenous leukemia, acute lymphoblastic leukemia, chroniclymphocytic leukemia, and blood cancer, but is not limited thereto.

The degenerative neurological disease can be any one selected from thegroup consisting of stroke, paralysis, memory loss, memory impairment,dementia, forgetfulness, Parkinson's disease, Alzheimer's disease,Pick's disease, Creutzfeld-Kacob disease, Huntington's disease andamyotrophic lateral sclerosis, but is not limited thereto.

Furthermore, the present invention provides a health functional food forpreventing or improving heat shock protein 90 (HSP90)-mediated diseasecomprising a benzamide compound represented by following ChemicalFormula 2 or a pharmaceutically acceptable salt thereof, as an activeingredient:

wherein R₁ is halogen or C1-C4 alkyl,

R₂ is

each of R₃ and R₄ can be the same as or different from to each other andis any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy,

each of R₅ and R₆ can be the same as or different from to each other andis any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy, or R₅ and R₆ are connected to each other to form 5- or6-membered ring,

R₇ is any one selected from the group consisting of H, C1-C4 alkyl andC1-C4 alkoxy,

R₈ is H or halogen,

R₉ is any one selected from the group consisting of C1-C4 alkyl C1-C4alkoxy and benzyl groups.

In the benzamide compound represented by the Chemical Formula 2, R₁ canbe halogen or C1-C4 alkyl, and R₂ can be

R₃ can be any one selected from the group consisting of H, C1-C4 alkyland C1-C4 alkoxy, and R₉ is any one selected from the group consistingof C1-C4 alkyl, C1-C4 alkoxy and benzyl groups.

The benzamide compound represented by the Chemical Formula 2 can be anyone selected from the group consisting ofN-benzyl-5-chloro-2,4-dihydroxybenzamide,N-benzyl-5-chloro-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-(4-methoxybenzyl)-N-methylbenzamide,5-chloro-N-(3,4-dimethoxybenzyl)-2,4-dihydroxy-N-methylbenzamide,N-(benzo[d][1,3]dioxol-5-ylmethyl)-5-chloro-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(3,4,5-trimethoxybenzyl)benzamide,5-chloro-N-(2-chloro-3,4,5-trimethoxybenzyl)-2,4-dihydroxy-N-methylbenzamide,N-(2-bromo-3,4,5-trimethoxybenzyl)-5-chloro-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(3-methylbenzyl)benzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(2-methylbenzyl)benzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(4-methylbenzyl)benzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(4-(methylcarbamoyl)benzyl)benzamide,5-chloro-N-(4-(ethylcarbamoyl)benzyl)-2,4-dihydroxy-N-methylbenzamide,5-chloro-2,4-dihydroxy-N-methyl-N-(4-(propylcarbamoyl)benzyl)benzamide,2,4-dihydroxy-5-isopropyl-N-methyl-N-(4-(methylcarbamoyl)benzyl)benzamide,N-(4-(ethylcarbamoyl)benzyl)-2,4-dihydroxy-5-isopropyl-N-methylbenzamide,2,4-dihydroxy-5-isopropyl-N-methyl-N-(4-(propylcarbamoyl)benzyl)benzamideand N-benzyl-2,4-dihydroxy-5-isopropyl-N-methylbenzamide, and preferably2,4-dihydroxy-5-isopropyl-N-methyl-N-(4-(propylcarbamoyl)benzyl)benzamide.

The pharmaceutical compositions according to the present invention mayfurther comprise suitable carriers, excipients or diluentsconventionally used in the manufacture of pharmaceutical compositions.

Examples of carriers, excipients or diluents which can be used in thepresent invention include lactose, dextrose, sucrose, sorbitol,mannitol, xylitol, erythritol, maltitol, starch, acacia rubber,alginate, gelatin, calcium phosphate, calcium silicate, cellulose,methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone,water, methythydroxybenzoate, propylhydroxybenzoate, talc, magnesiumstearate or mineral oil, etc.

The pharmaceutical composition according to the present invention may beformulated as oral preparations such as powders, granules, tablets,capsules, suspensions, emulsions, syrups, aerosols or the like,externals, suppositories and sterilized injection solutions according toa conventional method.

In the case of formulation, a diluent or excipient such as commonly usedfiller, an extender, a binder, a wetting agent, a disintegrant, asurfactant or the like is used. Solid formulations for oraladministration include tablets, pills, powders, granules, capsules,etc., which may contain at least one excipient such as starch, calciumcarbonate, sucrose, lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearateand talc are also used. Examples of the liquid preparation for oraladministration include suspensions, internal solutions, emulsions,syrups and the like and various excipients such as wetting agents,sweeteners, fragrances and preservatives may be included in addition towater and liquid paraffin which are commonly used simple diluents.

Formulations for parenteral administration include sterile aqueoussolutions, non-aqueous solutions, suspensions, emulsions, freeze-driedpreparations, and suppositories. Examples of the non-aqueous solutionsor suspending agent include propylene glycol, polyethylene glycol,vegetable oil such as olive oil, injectable ester such as ethyl oleate,and the like. Witepsol, macrogol, tween 61, cacao paper, laurin,glycerogelatin and the like can be used, as the base of suppositories.

Further, the dosage of the pharmaceutical composition according to thepresent invention may be increased or decreased depending on the routeof administration, degree of disease, sex, weight, age, and the like.Accordingly, the dosage amounts do not in any way limit the scope of theinvention.

The pharmaceutical composition may be administered to mammals such asrats, mice, livestock, humans, and the like in a various routes. Allmodes of administration may be expected, for example, by oral, rectal orintravenous, intramuscular, subcutaneous, intratracheal, intrauterine,or intracerebroventricular injections.

The dihydroxyphenyl-based compound or the benzamide compound of thepresent invention can be used in the form of a pharmaceuticallyacceptable salt and an acid addition salt formed by a pharmaceuticallyacceptable free acid is useful as the salt. As the free acid, inorganicacid and organic acid can be used. As the inorganic acid, hydrochloricacid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc.can be used and as the organic acid, citric acid, acetic acid, maleicacid, fumaric acid, glycolic acid, methanesulfonic acid, acetic acid,glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid,galacturonic acid, embonic acid, glutamic acid, citric acid and asparticacid. Preferably, hydrochloric acid is used as the inorganic acid, andmethanesulfonic acid is used as the organic acid.

In addition, the dihydroxyphenyl-based compound or benzamide compound ofthe present invention includes not only pharmaceutically acceptablesalts, but also all salts, hydrates and solvates which can be preparedby a conventional method.

The addition salt according to the present invention can be prepared bya conventional method and for example, by dissolving thedihydroxyphenyl-based compound of Chemical Formula 1 in a water-miscibleorganic solvent such as acetone, methanol, ethanol, acetonitrile or thelike and adding an organic acid or by adding an aqueous acid solution ofan inorganic acid, followed by precipitation or crystallization.Subsequently, a solvent or an excess acid may be evaporated from themixture and then dried to obtain an additional salt or the precipitatedsalt may be suction filtrated.

The health functional food according to the present invention may beprovided in the form of powder, granule, tablet, capsule, syrup orbeverage. Other food or food additives can be used together in additionto the health functional food may be a compound represented by the aboveChemical Formula 1 or 2 and can be suitably used according to aconventional method. The amount of the active ingredient to be mixed canbe appropriately determined according to its use purpose, for example,prevention, health or therapeutic treatment.

The compound represented by Chemical Formula 1 or 2 contained in thehealth functional food may be used in accordance with the effective doseof the pharmaceutical composition but may be used for health and hygienepurposes or for a long period. It may be less than the above range, andsince the active ingredient has no problem in terms of safety, it can beused in an amount exceeding the above range.

There is no particular limitation on the kind of the health functionalfood. Examples of the health functional food include meat, sausage,bread, chocolate, candy, snack, confectionery, pizza, ramen, othernoodles, gums, dairy products including ice cream, soup, beverage, tea,drinks, an alcoholic beverage, and a vitamin complex.

Hereinafter, the present invention will be described in detail withreference to the following examples. It should be noted, however, thatthe following examples are illustrative of the present invention and arenot intended to limit the scope of the present invention. Embodiments ofthe present invention are provided to more fully describe the presentinvention to those skilled in the art.

I. Synthesis of Dihydroxyphenyl Compounds and Evaluation of BiologicalActivity REFERENCE EXAMPLE 1

1. Reagents and Laboratory Equipment

All reagents and solvents were purchased from the manufacturer and usedwithout further purification.

All experiments dealing with moisture sensitive compounds were performedunder an argon atmosphere.

Concentration or solvent removal was carried out using a rotaryevaporator under reduced pressure.

Analytical thin layer chromatography was performed on precoated silicagel F254 TLC plates (silica gel F254 TLC plates, E, Merck) and UV lightwas visualized by staining with iodine gas.

Column chromatography can be performed on medium pressure on silica(Merck Silica gel 40-63 μm) or in Biotage SP1 flash purification system(Biotage SP1) using prepacked silica gel cartridges (Biotage).

NMR analysis was carried out using ARX-300 (300 MHz or more)manufactured by Bruker.

Chemical shifts were recorded in per million (δ). The deuterium locksignal of the sample solvent was used as a reference, and the couplingconstants (J) were recorded in hertz (Hz).

The splitting pattern abbreviations are: s, singlet; d, doublet; t,triplet; q, quartet, dd, doublet of doublets; m, multiplet.

2. Cell Culture

Sk-Br3 (Korean cell line bank), a breast cancer cell, and H1975 cell(ATCC), a nonsmall cell lung cancer, were cultured in RPMI 1640containing 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) and RPMI 1640 containing L-glutamine (containing streptomycin (500mg/mL), penicillin (100 units/mL) and 10% fetal bovine serum (FBS)).

The cells were cultured under a humidified atmosphere of 37° C. and 5%CO₂.

EXAMPLE 1 Synthesis of Dihydroxyphenyl Compound

Compounds 5 to 9, Compounds 10a to 10i and Compounds 11a to 11j weresynthesized in the manner as Reaction Scheme 1 below.

2,4-dihydroxybenzoic acid (5) was treated with sulfuric acid in methanolto obtain 2,4-dihydroxybenzoate (6) at a yield of 99%.

The chlorination of 2,4-dihydroxybenzoate (6) and sulfuryl chlorideleads to the formation of chlorinated product (7) and its region-isomer,methyl 3-chloro-2,4-dihydroxybenzoate in a molar ratio of 10:1. Theformed region-isomer was removed by silica gel chromatography carefully.

Compound 7 was protected with an allyl bromide in the presence ofpotassium carbonate to obtain an allyl-protected ester 8 in a yield of99%.

Thereafter, the ester 8 was converted to the carboxylic acid 9 in ayield of 97% using water and sodium hydroxide in methanol.

In addition, the amide coupling reaction between the carboxylic acid 9and various amines is carried out in the presence ofdiisopropylethylamine (DIPEA) in ethylene dichloride (EDC),hydroxybenzotriazole (HOBt) or dimethylformamide (DMF) to obtain amides10a to 10j.

Finally, the aryl-protected group was removed using PdCl₂(PPh₃)₂ andammonium formate under microwave irradiation to obtain Compounds 11a to11j.

Compound 11a, 11d and 11g to 11i were obtained using theabove-synthesized carboxylic acid ester 7.

The carboxylic acid ester 7 was converted to carboxylic acid 18 usingsodium hydroxide in methanol without aryl-protection.

The Friedel-Crafts alkylation of methyl 2,4-dihydroxybenzoate (6) wascarried out together with isopropyl bromide and aluminum chloride.

Compound 12 was protected with aryl bromide in the presence of potassiumcarbonate to produce ester 13, which was converted using sodiumhydroxide to obtain carboxylic acid 14 in a yield 58%.

The amide coupling reaction of the carboxylic acid 14 with1-methyl-3-phenylpiperazine (15) was carried out in the presence ofDIPEA in EDC, HOBt or DMF to obtain an amide 16.

Finally, the aryl-protected group was removed using PdCl₂(PPh₃)₂ andammonium formate under microwave irradiation to obtain Compound 17.

1. Methyl 2,4-dihydroxybenzoate (6)

2,4-dihydroxybenzoic acid (10.2 g, 66.0 mmol) and sulfuric acid (5 mL)in methanol (MeOH, 40 mL) were stirred under argon with refluxingcondenser at 100° C. for 12 hours. The mixture was cooled to roomtemperature, concentrated under pressure and poured into 40 mL of H₂O inan ice bath.

The resulting white solid was dissolved in ethyl acetate and then washedwith saturated NaHCO₃ solution to filter it. The organic layer was driedover Na₂SO₄ and pressure seas applied to produce Compound 6 in a yieldof 86%.

R_(f)=0.20 (2:8 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 10.97(s, 1H), 7.74 (d, J=8.8 Hz, 1H), 6.40 (d, J=2.0 Hz, 1H), 6.37 (dd, J=8.8Hz, 2.4 Hz, 1H), 5.36 (s, 1H), 3.91 (s, 1H).

2. Methyl-5-chloro-2,4-dihydroxybenzoate (7)

Compound 6 (10.0 g, 59.5 mmol) and sulfonyl chloride (4.30 mL, 59.5mmol) in methylene chloride (CH₂Cl₂) were stirred at 0° C. for 24 hoursunder argon.

The mixture was neutralized with 10% NaOH to pH 5, concentrated underpressure and then extracted with ethyl acetate.

The organic layer was washed three times with saturated NaHCO₃ solution,dried over Na₂SO₄ and concentrated under pressure to obtain Compound 7in a yield of 45%.

R_(f)=0.26 (2:8 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 10.84(s, 1H), 7.81 (s, 1H), 6.61 (s, 1H), 6.00 (s, 1H), 3.92 (s, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 169.6, 162.5, 157.3, 130.3, 111.5, 106.8, 104.3, 52.6

3. Methyl 2,4-bis(allyloxy)-5-chlorobenzoate (8)

A mixture of Compound 7 (6.09 g, 30.00 mmol), acyl bromide (6.75 ml,78.02 mmol) and potassium carbonate (10.78 mL, 78.02 mmol) in DMF wasstirred under argon at room temperature for 24 hours. The mixture wasconcentrated under pressure and extracted with ethyl acetate.

The organic layer was washed three times with saturated NaHCO₃ solution,dried over Na₂SO₄ and concentrated under pressure to obtain Compound 8in a yield of 100%.

R_(f)=0.30 (2:8 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 7.80(s, 1H), 6.39 (s, 1H), 5.99-5.91 (m, 2H), 5.45 (dd, J=17.2 Hz, 1.2 Hz,1H), 5.39 (dd, J=17.2 Hz, 0.8 Hz, 1H), 5.25 (d, J=8.0 Hz, 1H), 5.23 (d,J=8.4 Hz, 1H), 4.51 (dd, J=14.8 Hz, 5.2 Hz, 4H), 3.77 (s, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 164.7, 158.7, 157.7, 133.0, 132.2, 131.7, 118.1,117.4, 113.9, 112.6, 99.4, 69.7, 51.6

4. 2,4-bis(allyloxy)-5-chlorobenzoic Acid (9)

Compound 8 (9.43 g. 37.99 mmol) and 25 ml of methanol-25 ml of sodiumhydroxide in H₂O (5 g, 10%) were stirred at room temperature for 30hours. The mixture was neutralized with 1N HCl to pH 6 and extractedthree times with ethyl acetate.

The organic layer was dried over Na₂SO₄ and concentrated under pressureto obtain Compound 9 in a yield of 80%.

R_(f)=0.11 (4:6 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 8.16(s,1H), 6.54(s, 1H), 6.10-5.99(m, 2H), 5.51-5.35(m, 2H), 4.76-4.65(m, 4H).

5. (2,4-bis(allyloxy)-5-chlorophenyl)(pyrrolidin-1-yl)methanone (10a)

Compound 9 (0.36 g. 11.33 mmol), pyrrolidine (0.13 ml, 1.60 mmol),N,N′-dicyclohexylcarbodiimide (0.41 g, 2.00 mmol),1-hydroxybenzotriazole (0.22 g, 1.60 mmol) and N,N-diisopropylethylamine(0.23 mL, 1.33 mmol) were dissolved in 4 ml of DMF and stirred at 120°C. for 3 hours under microwave irradiation (using Biotage Initiator).

The mixture was dissolved in ethyl acetate and the organic layer waswashed with water, dried over Na₂SO₄ and concentrated under pressure.Thereafter, Compound 10a was obtained in a yield of 65% using MPLC(Biotage SNAP HP-Sil column).

R_(f)=(3:7 ethyl acetate:hexane). ¹H NMR (400 MHz, MeOD) δ 7.26 (s, 1H),6.77 (s, 1H), 6.15-6.02 (m, 2H), 5.51 (d, J=17.2 Hz, 1H), 5.43 (d,J=17.2 Hz, 1H), 5.32 (t, J=11.2 Hz, 11.2 Hz, 2H), 4.68 (dd, J=17.2 Hz,4.4 Hz, 4H), 3.70 (t, J=6.8 Hz, 6.8 Hz, 2H), 3.33 (s, 2H), 2.02-1.89 (m,4H). ¹³C NMR (100 MHz, MeOD) δ 167.2, 155.7, 154.2, 132.8, 132.6, 128.4,119.8, 116.7, 116.4, 114.1, 99.41, 69.4, 69.2, 45.7, 33.4, 25.4, 24.2

6 (2,4-bis(allyloxy)-5-chlorophenyl)(piperidin-1-yl)methanone (10b)

Compound 9 (0.30 g, 1.12 mmol), piperidine (0.12 ml, 1.23 mmol),N,N′-dicyclohexylcarbodiimide (0.46 g, 2.23 mmol),1-hydroxybenzotriazole (0.15 g, 1.12 mmol) and N,N-diisopropylethylamine(0.19 mL, 1.12 mmol) were dissolved in 4 ml of DMF, and stirred at 120°C. for 3 hours under microwave irradiation (Biotage Initiator).

The mixture was dissolved in ethyl acetate and the organic layer waswashed with water, dried over Na₂SO₄ and concentrated under pressure.Thereafter, Compound 10b was obtained in a yield of 44% using MPLC(Biotage SNAP HP-Sil column).

R_(f)=0.21 (3:7 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 7.23(s, 1H), 6.67 (s, 1H), 6.06-5.92 (m, 2H), 5.44 (d, J=17.6 Hz, 1H), 5.37(d, J=17.2 Hz, 1H), 5.31 (d, J=10.8 Hz, 1H), 5.26 (d, J=10.4 Hz, 1H),5.59 (d, J=4.8 Hz, 2H), 4.52 (t, J=4.4 Hz, 5.2 Hz, 2H), 3.66 (dd, J=17.2Hz, 1.6 Hz, 1H), 3.19 (dd, J=12.0 Hz, 7.6 Hz, 2H), 1.614 (s, 4H), 1.48(d, J=42.8 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 166.3, 155.3, 154.0,132.8, 132.5, 129.4, 120.3, 118.3, 117.9, 115.2, 99.9, 70.2, 69.8, 48.2,42.9, 26.5, 25.8, 24.7.

7. (2,4-bis(allyloxy)-5-chlorophenyl)(morpholino) methanone (10c)

Compound 9 (0.50 g, 1.85 mmol), morpholine (0.18 ml 2.04 mmol),N,N′-dicyclohexylcarbodiimid (0.76 g, 3.70 mmol), 1-hydroxybenzotriazole(0.55 g, 1.85 mmol) and N,N-diisopropylethylamine (0.32 mL, 1.85 mmol)were dissolved in 4 mL of DMF and stirred at 120° C. for hours undermicrowave irradiation.

The mixture was dissolved in ethyl acetate and the organic layer waswashed water, dried over Na₂SO₄, and concentrated under pressure.Thereafter, Compound 10c was obtained in a yield of 68% using MPLC(Biotage SNAP HP-Sil column).

R_(f)=0.20 (4:6 ethyl acetate:hexane). R_(f)=0.21 (3:7 ethylacetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 7.27 (s, 1H), 6.47 (s, 1H),6.07-5.93 (m, 2H), 5.44 (dd, J=17.2 Hz, 1.2 Hz, 1H), 5.36 (dd, J=17.2Hz, 1.6 Hz, 1H), 5.32 (dd, J=4 Hz, 2.8 Hz, 2H), 5.29 (dd, J=10.4 Hz, 1.2Hz, 2H), 4.56 (dd, J=30.8 Hz, 4.0 Hz, 4H).

8. (2,4-bis(allyloxy)-5-chlorophenyl)(4-methylpiperazin-1-yl)methanone10e)

Compound 9 (0.30 g, 1.12 mmol), 1-methylpiperazine (0.19 ml, 1.67 mmol),N,N′-dicyclohexylcarbodiimide (0.46 g, 2.23 mmol) 1-hydroxybenzotriazole(0.15 g, 1.12 mmol) and N,N-diisopropylethylamine (0.19 mL, 1.12 mmol)were dissolved in 4 mL of DMF and stirred at 120° C. for 3 hours undermicrowave irradiation (Biotage Initiator).

The mixture was dissolved in ethyl acetate and the organic layer waswashed with water, dried over Na₂SO₄ and concentrated under pressure.Thereafter Compound 10e was obtained in a yield of 64% using MPLC(Biotage SNAP HP-Sil column).

R_(f)=0.14 (9:1 ethyl acetate:methanol). ¹H NMR (400 MHz, CDCl₃) δ 7.24(s, 1H), 6.46 (s, 1H), 6.06-5.90 (m, 2H), 5.43 (dd, J=17.2 Hz, 1.2 Hz,1H), 5.34 (dd, J=17.2 Hz, 1.2 Hz, 1H), 5.30 (dd, J=10.4 Hz, 0.8 Hz, 1H),5.25 (dd, J=10.4 Hz, 1.2 Hz, 1H), 4.58 (d, J=4 Hz, 2H), 4.50 (s, 2H),3.76 (d, J=61.6 Hz, 2H), 3.27 (d, J=15.2 Hz, 2H), 2.27 (d, J=35.2 Hz,4H), 2.27 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 166.4, 155.5, 154.0,132.5, 132.4, 129.6, 119.4, 118.3, 118.1, 115.2, 99.6, 70.1, 69.8, 55.3,54.8, 46.9, 46.2, 41.8

9. 1-(4-(2,4-bis(allyloxy)-5-chlorophenyl)piperazin-1-yl)ethanone (10f)

Compound 9 (0.30 g, 1.12 mmol), 1-ethylpiperazine (0.21 ml, 1.67 mmol),N,N′-dicyclohexylcarbodiimide (0.46 g, 2.23 mmol) 1-hydro benzotriazole(0.15 g, 1.12 mmol) and N,N-diisopropylethylamine (0.19 mL, 1.12 mmol)were dissolved in 4 mL, of DMF and stirred at 120° C. for 3 hours undermicrowave irradiation (Biotage Initiator).

The mixture was dissolved in ethyl acetate and the organic laser waswashed with water, dried over Na₂SO₄ and concentrated under pressure.Thereafter, Compound 10f was obtained in a yield of 86% using MPLC(Biotage SNAP HP-Sil column).

R_(f)=0.20 (9:1 ethyl acetate:methanol). ¹H NMR (400 MHz, CDCl₃) δ 7.25(s, 1H), 6.46 (s, 1H), 6.04-5.93 (m, 2H), 5.42 (dd, J=17.2 Hz, 1.2 Hz,1H), 5.32 (d, J=15.1 Hz, 1H), 5.26 (d, J=14.4 Hz, 2H), 4.54 (d, J=32.8Hz, 2H), 3.77-3.18 (m, 8H), 2.07 (d, J=23.6 Hz, 3H).

10. (5-chloro-2,4-dihydroxyphenyl) (pyrrolidin-1-yl)methanone (11a)

Compound 10a (0.23 g, 0.72 mmol) was stirred in the presence ofPdCl₂(PPh₃)₂ (23 mg) and 4 ml of ammonium formate in THF (150 mg) at120° C. for 30 min under microwave irradiation.

The reaction mixture was diluted with ethyl acetate and the organiclayer was washed with water. It was dried over Na₂SO₄, concentratedunder pressure to obtain Compound 11a by using MPLC in yield of 29%.

R_(f)=0.26 (3:7 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 7.20(s, 1H), 6.29 (s, 1H), 4.24 (s, 2H), 3.15 (t, J=1.6 Hz, 1.6 Hz, 4H),1.77 (t, J=6.4 Hz, 6.8 Hz, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 169.0, 158.9,156.3, 129.4, 111.5, 110.9, 104.2, 49.3, 49.1, 48.3, 48.1.

11. (5-chloro-2,4-dihydroxyphenyl) (piperidin-1-methanone (11b)

Compound 10h (0.17 g, 0.50 mmol) seas stirred in the presence ofPdCl₂(PPh₃)₂ (17 mg) and 4 ml of ammonium formate in THF (150 mg) at120° C. for 30 minutes under microwave irradiation.

The reaction mixture was diluted with ethyl acetate and the organiclayer was washed with water. It was dried over Na₂SO₄, concentratedunder pressure and then Compound 11h was obtained in a yield of 28%using MPLC.

R_(f)=0.27 (5:5 ethyl acetate:hexane). ¹H NMR (400 MHz, MeOD) δ 7.04 (s,1H), 6.42 (s, 1H), 3.45 (s, 4H), 1.62 (d, J=4.8 Hz, 2H), 1.55 (d, J=4.4Hz, 4H). ¹³C NMR (100 MHz, MeOD) δ 169.2, 156.0, 154.9, 129.8, 116.9,112.2, 104.4, 36.8, 34.5, 26.8, 25.2

12. (5-chloro-2,4-dihydroxyphenyl) (morpholino)methanone (11c)

Compound 10c (0.42 g, 1.25 mmol) was stirred in the presence ofPdCl₂(PPh₃)₂ (42 g) and 4 ml of ammonium formate in THF (150 mg) at 120°C. for 30 minutes under microwave irradiation.

The reaction mixture was diluted with ethyl acetate and the organiclayer was washed with water. It was dried over Na₂SO₄, concentratedunder pressure and then Compound 11c was obtained in a yield of 49%using MPLC.

R_(f)=0.24 (4:6 ethyl acetate:hexane). ¹H NMR (400 MHz, MeOD) δ 7.16 (s,1H), 6.48 (s, 1H), 3.69 (d, J=4.0 Hz, 4H), 3.57 (s, 4H)

13. (5-chloro-2,4-dihydroxyphenyl)(4-methylpiperazin-1-yl)methanone(11e)

Compound 10e (0.25 g, 0.72 mmol) was stirred in the presence ofPdCl₂(PPh₃)₂ (25 mg) and 4 ml of ammonium formate in THF (150 mg) at120° C. for 30 minutes under microwave irradiation.

The reaction mixture was diluted with ethyl acetate and the organiclayer was washed with water. It was dried over Na₂SO₄, concentratedlender pressure and then Compound 11e was obtained in two steps in ayield of 30% using MPLC.

R_(f)=0.20 (7:3 ethyl acetate:Methanol). ¹H NMR (400 MHz, MeOD) δ 7.13(s, 1H), 6.47 (s, 1H), 3.59 (d, J=8.0 Hz, 6H), 3.52 (s, 2H) 2.10 (s,3H). ¹³C NMR (125 MHz, DMSO) δ 165.9, 154.4, 153.4, 128.9, 116.0, 109.9,103.5, 54.5, 45.6, 40.4

14. 1-(4-(5-chloro-2,4-dihydroxybenzol)piperazin-1-yl)ethanone (11f)

Compound 10f (0.17 g, 0.45 mmol)) stirred in the presence ofPdCl₂(PPh₃)₂ (17 mg) and 4 ml of ammonium formate in THF (150 mg) at120° C. for 30 minutes under microwave irradiation.

The reaction mixture was diluted with ethyl acetate and the organiclayer was washed with water. It was dried over Na₂SO₄, concentratedunder pressure and then compound 11f was obtained in two steps in ayield of 61% using MPLC.

R_(f)=0.14 (9:1 ethyl acetate:Methanol). ¹H NMR (400 MHz, MeOD) δ 7.14(s, 1H), 6.46 (s, 1H), 3.60 (s, 4H), 2.51 (t, J=4.8 Hz, 4.8 Hz, 4H),2.35 (s, 3H). ¹³C NMR (125 MHz, DMSO) δ 168.4, 166.2, 154.5, 153.4,129.2, 115.9, 110.1, 103.5, 45.7, 40.9, 21.2

15. 5-chloro-2,4-dihydroxybenzoic Acid (18)

Compound 7 (1.98 g, 9.76 mmol) and 30 ml of methanol-30 ml of sodiumhydroxide in H₂O (6 g) were stirred at room temperature for 24 hours.The mixture was neutralized with 3N HCl to pH 6 and extracted threetimes with ethyl acetate.

The organic layer was dried over Na₂SO₄ and concentrated under reducedpressure to obtain Compound 18 in a yield of 100%.

¹H NMR (400 MHz, CDCl₃) δ 7.75 (s, 1H), 6.40 (s, 1H). ¹³C NMR (100 MHz,CDCl₃) δ 171.5, 162.0, 158.9, 131.5, 112.1, 105.8, 103.6.

16. (5-chloro-2,4-dihydroxyphenyl) (piperazin-1-yl)methanone (11d)

Compound 18 (0.21 g, 1.13 mmol), test-butyl 1-piperazinecarboxylate(0.32 g, 1.69 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.46g, 2.26 mmol), N,N′-dicyclohexylcarbodiimide (0.46 g, 2.23 mmol), and1-hydroxybenzotriazole (0.15 g, 1.13 mmol) and N,N-diisopropylethylamine(0.20 mL, 1.13 mmol) were dissolved in 4 ml of DMF, and stirred at 120°C. for 3 hours under microwave irradiation.

The mixture was dissolved in ethyl acetate and the organic layer waswashed with 1 N—HCl solution, dried over Na₂SO₄, and concentrated underpressure. Thereafter, an intermediate compound was synthesized in ayield of 69% using MPLC, (Biotage SNAP HP-Sil column).

R_(f)=0.23 (5:5 ethyl acetate:hexane).

The intermediate compound was stirred for 24 hours at room temperatureire the presence of 10 ml of 6N HCl solution and 10 ml of THF. Afterdiluting the reaction with ethyl acetate, the organic layer was washedwith water, dried over Na₂SO₄, and concentrated under pressure. It aspurified by MPLC to obtain Compound 11d in two steps in a yield of 32%.

¹H NMR (400 MHz, MeOD) δ 7.13 (s, 1H), 6.45 (s, 1H), 6.45 (s, 1H), 6.74(s, 4H), 3.21 (d, J=4.4 Hz, 4H). ¹³C NMR (100 MHz, MeOD) δ 169.7, 157.1,155.2, 131.0, 115.5, 113.2, 104.7, 62.9, 44.7

17. 4-(5-chloro-2,4-dihydroxybenzoyl)piperazin-2-one (11 g)

Compound 18 (0.30 g, 1.12 mmol), piperidine (0.12 ml, 1.23 mmol),N,N′-dicyclohexylcarbodiimide (0.46 g, 2.23 mmol),1-hydroxybenzotriazole (0.15 g, 1.12 mmol) and N,N-diisopropylethylamine(0.19 mL, 1.12 mmol) were dissolved in 4 ml of DMF and stirred at 120°C. for 3 hours under microwave irradiation.

The reaction mixture was diluted with ethyl acetate and the organiclayer was washed with water. It was dried over Na₂SO₄, concentratedunder pressure and Compound 11g was obtained in a yield of 44% usingMPLC.

R_(f)=0.21 (3:7 ethyl acetate:hexane). ¹H NMR (400 MHz, DMSO) δ 10.07(s, 1H), 8.04 (s, 1H), 7.09 (s, 1H), 6.55 (s, 1H), 3.92 (s, 2H), 3.53(s, 2H), 3.36 (s, 3H), 3.19 (s, 2H)

18. 1-(5-chloro-2,4-dihydroxybenzoyl) pyrrolidin-3-one (11h)

Compound 18 (0.05 g, 0.27 mmol), 3-pyrrolidinone hydrochloride (0.05 g,0.41 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.11 g, 0.55mmol), 1-hydroxybenzotriazole (0.04 g, 0.27 mmol),N,N′-diisopropylethylamine (0.20 mL, 1.13 mmol) andN,N′-dicyclohexylcarbodiimide (0.10 mL, 0.55 mmol) were dissolved in 4 mof DMF and stirred at 120° C. for 3 hours under microwave irradiation(Biotage Initiator).

The organic layer was washed with 1N—HCl solution, dried over Na₂SO₄,concentrated under pressure and purified by MPLC to obtain Compound 11hin a yield of 10%.

R_(f)=0.21 (5:5 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 11.04(s, 1H), 7.39 (s, 1H), 6.66 (s, 1H), 5.93 (s, 1H), 4.21 (t, J=8.0 Hz,7.6 Hz, 2H), 4.14 (s, 2H), 2.67 (t, J=7.6 Hz, 8.0 Hz, 2H).

19. 1-(5-chloro-2,4-dihydroxybenzoyl)piperidin-4-one (11i)

Compound 18 (0.20 g, 1.09 mmol), 4-piperidine (0.25 g, 1.63 mmol),1-ethyl-3-(3-dimethylaminopropyl) (0.42 g, 2.17 mmol),1-hydroxybenzotriazole (0.15 g, 1.09 mmol) and N,N-diisopropylethylamine(0.39 mL, 2.17 mmol) wore dissolved in 4 ml of DMF and stirred at 120°C., for 3 hours under a microwave irradiation (Biotage Initiator).

The reaction mixture was diluted with ethyl acetate and the organiclayer was washed with 1 N HCl solution. It was dried over Na₂SO₄,concentrated under pressure and then Compound 11i was obtained in ayield of 3% by using MPLC.

R_(f)=0.21 6:4 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 10.14(s, 1H), 7.31 (s, 1H), 6.67 (s, 1H), 5.88 (s, 1H), 3.97 (t, J=6.4 Hz,6.0 Hz, 4H), 2.58 (t, J=6.0 Hz, 6.4 Hz, 4H).

20.(5-chloro-2,4-dihydroxyphenyl)(4-methyl-2-phenylpiperazin-1-yl)methanone(11j)

Compound 18 (0.20 g, 1.05 mmol), 1-methyl-3-phenylpiperazine (0.28 g,1.58 mmol), 1-ethyl-3-(3-dimethylaminopropyl) (0.40 g, 2.10 mmol),1-hydroxybenzotriazole (0.14 g, 1.05 mmol) and N,N-diisopropylethylamine(0.19 mL, 1.05 mmol) were dissolved in 4 ml of DMF and stirred at 120°C. for 3 hours under a microwave irradiation (Biotage Initiator).

The reaction mixture was diluted with ethyl acetate and the organiclayer was washed with 1 N HCl solution. It was dried over Na₂SO₄,concentrated under pressure and purified by MPLC to obtain Compound 11jin a yield of 23%.

R_(f)=0.21 (8:2 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 7.46(d, J=4.4 Hz, 2H), 7.37 (t, J=7.2 Hz, 7.2 Hz, 2H), 7.27 (t, J=7.2 Hz,6.8 Hz, 1H), 7.17 (s, 1H), 6.61 (s, 1H), 5.58 (s, 1H), 4.24 (s, 1H),3.24 (d, J=12.0 Hz, 1H), 3.22 (t, J=12.4 Hz, 10.4 Hz, 1H), 2.81 (d,J=10.8 Hz, 1H), 2.50 (dd, J=12.0 Hz, 4.0 Hz, 1H), 2.32 (s, 3H),2.21-2.14 (m, 1H).

21. Methyl 2,4-dihydroxy-5-isopropylbenzoate (12)

Compound 6 (3.9 g, 23.0 mmol), 2-bromopropane (4.3 mL, 46.0 mmol) andaluminum chloride (6.1 g, 46.0 mmol) were dissolved in CH₂Cl₂ and thenstirred under argon at 50° C. for 24 hours with a reflux condenser undera microwave irradiation (Biotage Initiator).

3-Bromopropane (4.3 ml, 46.0 mmol) was added to the reaction mixturethree times every 6 hours.

The mixture was neutralized with 10% NaOH to pH 5, concentrated underpressure and extracted with ethyl acetate.

The organic layer was washed with saturated NaHCO₃ solution three times,dried over Na₂ SO₄, concentrated under pressure and purified by columnto obtain Compound 12 in a yield of 45%.

R_(f)=0.21 (1:4 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃) δ 10.8(s, 1H), 7.64 (s, 1H), 6.34 (s, 1H), 5.53 (s, 1H), 3.92 (s, 3H),3.15-3.08 (m, 1H), 1.25 (d, J=10.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ170.7, 161.6, 159.6, 128.1, 127.1, 105.7, 103.2, 52.2, 26.7, 22.8

22. Methyl 2,4-bis(allyloxy)-5-isopropylbenzoate (13)

Compound 12 (2.1 g, 10.3 mmol), allyl bromide (2.3 mL, 26.8 mmol) andpotassium carbonate (3.7 g, 26.8 mmol) were dissolved in DMF and stirredfor 18 hours.

The mixture was diluted with ethyl acetate and the organic layer waswashed with H₂O, dried over Na₂SO₄, and concentrated under pressure toobtain Compound 13 in a yield of 84%.

¹H NMR (400 MHz, CDCl₃): δ 7.71 (s, 1H), 6.41 (s, 1H), 6.10-5.97 (m,2H), 5.50 (dd, J=17.2 Hz, 1.6 Hz, 1H), 5.41 (dd, J=17.2 Hz, 1.2 Hz, 1H),5.27 (d, J=10.8 Hz, 2H), 4.56 (dd, J=9.6 Hz, 4.8 Hz, 4H), 3.84 (s, 1H),3.26-3.19 (m, 1H), 1.19 (d, J=6.8 Hz, 6H).

23. 2,4-bis(allyloxy)-5-isopropylbenzoic Acid (14)

Compound 13 (2.5 g, 8.6 mmol) and 30 ml of methanol-30 ml of sodiumhydroxide in H₂O (1.7 g, 43.1 mmol) were stirred at room temperature for24 hours.

The mixture was diluted with ethyl acetate and the organic layer waswashed with 3N HCl solution and dried over Na₂SO₄, concentrated underpressure and purified using a column to obtain Compound 14 in a yield of58%.

R_(f)=0.18 (1:4 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃): δ 7.97(s, 1H), 6.43 (s, 1H), 6.10-5.97 (m, 2H), 5.47-5.39 (m, 2H), 5.30 (d,J=10.8 Hz, 2H), 4.73 (d, J=5.6 Hz, 2H), 4.57 (d, J=4.8 Hz, 2H),3.26-3.19 (m, 1H), 1.18 (d, J=6.8 Hz, 6H).

24.(2,4-bis(allyloxy)-5-isopropylphenyl)(4-methyl-2-phenylpiperazin-1-yl)methanone(16)

Compound 14 (0.18 g, 0.66 mmol), 1-methyl-3-phenylpiperazine (0.18 g,0.99 mmol), 1-ethyl-3-(3-dimethylaminopropyl) (0.25 g, 1.33 mmol),1-hydroxybenzotriazole (0.09 g, 0.66 mmol) and N,N-diisopropylethylamine(0.09 mL, 0.66 mmol) were dissolved in 4 ml of DMF and stirred at 120°C. for 3 hours under a microwave irradiation (Biotage Initiator).

The reaction mixture was diluted with ethyl acetate and the organiclayer was washed with 1 N HCl solution. It was dried over Na₂SO₄,concentrated under pressure and purified by MPLC to obtain Compound 16in a yield of 92%.

R_(f)=0.24 (3:7 ethyl acetate:hexane).

25.(2,4-dihydroxy-5-isopropylphenyl)(4-methyl-2-phenylpiperazin-1-yl)methanone(17)

Compound 16 (0.26 g, 0.61 mmol) was stirred under microwave irradiationin the presence of PdCl₂(PPh₃)₂ (10 mg) and 4 ml of ammonium formate inTHF (227 ma) at 120° C. for 30 minutes.

The reaction mixture was diluted with ethyl acetate and the organiclayer was washed with water. It was dried over Na₂SO₄, concentratedunder pressure and then Compound 17 was obtained in two steps in a yieldof 15% using MPLC.

R_(f)=0.32 (8:2 ethyl acetate:Hexane). ¹H NMR (400 MHz, CDCl₃): 7.45 (d,J=7.6 Hz, 2H), 7.37 (t, J=7.2 Hz, 7.6 Hz, 2H), 7.25(t, J=7.8 Hz, 7.6 Hz,1H), 6.99 (s, 1H), 6.40 (s, 1H), 5.58 (s, 1H), 4.24 (s, 1H), 3.44 (t,J=8.8 Hz, 12.0 Hz, 1H), 3.30 (t, J=12.4 Hz, 10.4 Hz, 1H), 3.05-3.00(m,1H), 2.79 (d, J=10.8 Hz, 1H), 2.47 (dd, J=12.0 Hz, 4.0 Hz, 1H), 2.30 (s,3H), 2.22-2.16 (m, 1H), 0.95 (dd, J=20.0 Hz, 6.0 Hz, 6H). ¹³C NMR (100MHz, CDCl₃) δ 171.5, 158.1, 138.8, 128.9, 127.3, 127.1, 126.4, 109.1,103.9, 60.6, 55.4, 45.6, 26.1, 22.5, 21.1, 19.9, 14.3

EXAMPLE 2 Evaluation of Biological Activity of Dihydroxyphenyl Compound

1. Confirmation of HSP90 Inhibitory Effect

The HSP90 inhibitory effect of the dihydroxyphenyl compound synthesizedin the Example 1 was confirmed by a Fluorescence Polarization assay (FPassay),

HFB buffer (100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) pH 7.3, 2 M KCl, 1 M MgCl₂, 1 M Na₂MoO₄, 100% NP₄O), HSP90αN-terminal domain (2 μM) protein, FITC (fluoresceinisothiocyanate)-labeled geldanamycin (GA) inhibitor (500 nM) andCompounds at concentrations of (0.001, 0.01, 0.1, 0.5, 1, 5, 10, 50, 100μM) were added to each well.

Thereafter, plates were incubated at 4° C. for 14 hours. Thepolarization values of the millipolarization units were measured at anexcitation wavelength of 495 nm and an emission wavelength of 530 nm.

All experimental data were analyzed using Prism software (version 5.0,Graphpad Software, San Diego, Calif.). In addition, tPSA (TopologicalPolar Surface Area) calculated using Cherndraw software and the measuredvalues of Log P are shown in Tables 1 and 2 below.

Compounds 11a to 11i are represented by the Chemical Formula 3.

TABLE 1 HSP90 Inhibition Inhibition (FP) rate at 10 rate at 30 Entry RCompound (IC₅₀; μM) μM (%) μM (%) tPSA LogP 1

11a 0.348 8.1 22.1 60.7 1.13 2

11b 0.541 7.6 26.3 60.7 1.69 3

11c 0.383 24.3 44.0 70 0.66 4

11d 5.59 0 0 72.8 0.65 5

11e 0.253 26.3 48.7 64 1.23 6

11f 3.21 0 10.5 81.1 0.25 7

11g 1.37 0 0 89.9 0.45 8

11h 3.92 0 0 77.8 0.84 9

11i 0.670 12.5 48.2 77.8 0.6

Compounds 11j and 17 are represented by the above Chemical Formula 4.

TABLE 2 Inhibition Inhibition HSP90(FP) rate at 10 μM rate at 30 μMEntry R compound (IC₅₀; μM) (%) (%) tPSA LogP 10 Cl 11j 0.070 — — 64.013.22 11 (i-propyl) 17  0.0495 72.08 77.58 64.01 3.80

As a result, as shown in the Table 2, Compound 17 showed an IC₅₀ of0.0495 μM for HSP90 and an IC₅₀ of 98 μM for H1975.

In addition, HSP90 inhibitory activity of by Compound 17 confirmed by FPanalysis is shown in FIG. 1. The increased concentration of Compound 17added FITC-geladinomycin/HSP90α (N-terminal domain) protein response andrecorded FP readings.

As shown in the Table 2, Compound 17 showed HSP90 inhibitory activity ina concentration-dependent manner.

2. Confirmation of Cell Proliferation and Survival Inhibition

Next, non-metastatic lung cancer cells were treated with Compound 17 andthe cell proliferation rate or cell survival rate was measured by MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-1-2H-tetrazolium,inner salt] analysis.

Cells were dispensed in 96-well plates at 3000 cell/well (Sk-Br3) and2000 cell/well (H1975), and 100 μL of the medium was added per well andthen was incubated overnight.

The following day, Compound 17 at a concentration of 0, 0.01, 0.1, 1, 5,10, 30, 50 or 70 μM or 1% DMSO vehicle (control) was added to each well,and cultured at 37° C. for 1, 2 and 3 days.

Cell proliferation was evaluated using the Promega Cell Titer 96 AqueousOne Solution cell proliferation assay.

After compound and cell culture, 20 μL of assay substrate solution wasadded to each well and incubated at 37° C. for an additional 1 hour.

Cell proliferation was confirmed by measuring the absorbance at 490 nmusing a microplate reader (Tecan Infinite F200 Proplate reader).

Compound 17 at a concentration of 0, 0.01, 0.1, 1, 5, 10, 30, 50 or 70μM or 1% DMSO vehicle (control group) was added to each well andincubated at 37° C. for 72 hours and the absorbance was measured at 490nm and the measured value was expressed as a percentage of theabsorbance of the cells cultured only with DMSO to confirm cellsurvival.

As a result, as shown in FIG. 2, as time passed, the proliferation rateof the cells was inhibited in a concentration-dependent manner ofCompound 17 (FIG. 2A), and cell death was also observed (FIG. 2B).

3. Confirmation of H1975 Colony Formation Inhibitory Effect

H1975 cells were treated with Compound 17 at a concentration of 0.05 or0.5 μM for 3 weeks and then clogenic assay was performed.

First, RPMI1640 medium (10% FBS, 0.48% agar) was added to a 6-well plateand hardened, and then, 10000 cells (H1975) were dispensed in a mediumcontaining RPMI1640 medium (10% FBS, 0.33% agar) and solidified. Aftersolidification, the cells were treated with compound at a concentrationof 0.05 or 0.5 nM for 24 hours, followed by culturing for 3 weeks, andthen colony formation was confirmed using a crystal violet stainingsolution.

As a result, as shown in FIG. 3, Compound 17 inhibited the colonyformation of H1975 even at a low concentration of 0.05 M.

4. Protein Expression Analysis

Recombinant HSP90 was expressed from pET-15b plasmid (Novagen) intoEscherichia coli BL21 (DE3) cells (BioLabs).

The new colony was grown with shaking at a speed of 180 rpm until theabsorbance reached A₆₀₀=0.5 using LB broth medium containing 2.5 mg mL:(500 μl/200 ml) of ampicillin.

Thereafter, protein expression was induced byisopropyl-1-thio-β-D-galactopyranoside (final concentration 1 mM).

After decreasing the temperature to 18° C., the cells were incubatedovernight with shaking to obtain cell pellet by centrifugation (4000rpm, 20 min, 4° C.).

The cell pellet was suspended in NTA (nitrilotriacetate acid) buffer(containing 20 mM Tris pH 8.0, 0.5 M NaCl), sonicated in ice andcentrifuged to obtain supernatant (14000 rpm, 99 min, 4° C.).

The His-tagged protein was purified with a Histidine Trap column andnickel-nitriloacetic acid and separated by FPLC (Fast Protein LiquidChromatography: Phamacia).

To identify HSP proteins, proteins were analyzed by western blotting.

Salts were removed from the purified protein by the PD10 column andconcentrated to 2.4 mg/ml in Vivaspin 20 and stored at −70° C.

Next, the cells were dispensed into 100 mm culture dishes (1×10⁶/dish)and adhered to the bottom surface overnight. Compound 17 at aconcentration of 0.05, 0.1, 0.5 or 1 μM was added to the cells, followedby further culture for 24 hours.

In addition, cells were treated with DMSO (1%) or geladinamycin (1 μM)as a control and cultured for 24 hours.

The cultured cells were harvested and lysed in ice-cold lysis buffer (23mM Tris-HCl pH 7.6, 130 mM NaCl, 1% NP40, % sodium deoxycholate, 0.1%SDS (sodium dodecylsulfate)), and 30 μg of the lysate was separated bySDS-PAGE (SDS-polyacrylamide gel electrophoresis) and transferred to aPVDF membrane (Bio-Rad).

Membranes were blocked with TBST (Tween 20 containing iris-BufferedSaline) containing 5% skim milk and incubated with the respectiveprimary antibodies [EGFR (Epidermal growth factor receptor), Her2 (humanepidermal growth factor receptor type 2), Met, N-cadherin, E-cadherin,α-tubulin, acetyl-α-tubulin, protein kinase B (Akt), c-Raf, Cdk4(Cyclin-dependent kinase 4), HSP90, HSP70, PARP (Poly ADT-ribosePolymerase), caspase 3, cleaved caspase 3, truncated caspase 8, B-celllymphoma 2 (Bcl-2). Bax or β-Actin, purchased from Cell SignalingTechnology (USA)].

After binding to a secondary antibody conjugated with horseradishperoxidase, the protein was visualized by ECL (electrochemiluminescence,GE healthcare, USA).

As a result, as shown in FIG. 4, Compound 17 inhibited HSP protein inconcentration-dependent manner to confirm the degradation of HSP90client protein of EGFR, Her2, Met, Akt, c-Raf, Cpk4 and most of theclient proteins were denatured at 500 nM.

II. Synthesis of Benzarnide Compounds and Evaluation of BiologicalActivity REFERENCE EXAMPLE 2 Reagents and Experimental Apparatus

The reagents purchased from Sigma-Aldrich (St. Louis, Mo., USA), acrosorganics (Thermo Fisher Scientific In., Gell, Belgium), alfa aesar (AJohnson Malley Company, Karlsruhe, Germany) and Daejung (DaejeonghwageumCo., Kyunggi-do, Korea) were used. Compound synthesis was carried outunder argon or in atmosphere, and in some cases microwave (Biotage®,Uppsala, Sweden) was used. Extraction, recrystallization, columnchromatography and MPLC (medium pressure liquid column chromatography)were used to purify the product after synthesis. Silica gel 60(0.040-0.063 mm) was used as a filler for the column chromatography andSNAP cartridge (KP-Sil 25 g or KP-C18-HS 30 g) was used for MPLC.

Structure of each compound was identified using ¹H NMR and ¹³C NMRspectra by a Bruker spectrospin 400 spectrometer (Bruker co., Billerica,Mass., USA). CDCl₃, CD₃OD or dimethyl sulfoxide (DMSO)-d₆ was used as asolvent and the chemical shift value (δ) was expressed in ppm and thepeaks were expressed in d (doublet), t (triplet), m (multiplet) and dd(doublet of doublet).

A power supply unit (BioRad co., Hercules, Calif.), an image analyzer(Fuji, Tokyo, Japan) and a microplate reader (TECAN, Mannedorf,Switerland) were used for evaluating the biological activity.

EXAMPLE 3 Synthesis of Benzamide-Based Compound

Compounds 6a to 6h and 8a to 8c were synthesized in the same manner asin Reaction Scheme 4 below.

entry R compound  1

6a  2

6b  3

6c  4

6d  5

6e  6

6f  7

6g  8

6h  9

7a, 8a 10

7b, 8b 11

7c, 8c

1. Amine Synthesis Method I (6a-h)

One equivalent of each aldehyde and 1.5 equivalents of 40% methylaminein distilled water (d-water) were dissolved in MeOH and stirred at roomtemperature for 30 minutes. 0.5 equivalent of sodium borohydride (NaBH₄)was slowly added at 0° C. and then stirred for 1 hour. Thereafter, H₂Owas added to the mixture, MeOH was removed by distillation under reducedpressure, and extracted three times with dichloromethane (DCM). The DCMlayer was dried with NaSO₄ and filtered and the solvent was removed bydistillation under reduced pressure to obtain 6a-h in yields of32.0-76.4%.

2. Amine Synthesis Method II (8a-c)

One equivalent of benzoic acid, 1.5 equivalents 40% of methylamine ind-water, 2 equivalents of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) or DCC N,N-equivalent dicyclohexylcarbodiimide), 1equivalent of HOBt (hydroxybenzotriazole) and 1 equivalent of DIPEA(NN-diiso-propylethylamine) were dissolved in dimethylformamide (DMF)and microwave-reacted at 120° C. and 20 bar for 3 hours. Afterdissolving in ethyl acetate (EA) and washing several times with 1N HClsaturated aqueous solution, the EA layer was dried with Na₂SO₄ andfiltered, and the solvent was removed by distillation under reducedpressure. After purification by MINX or column chromatography, 1equivalent of the purified compound was dissolved in tetrahydrofuran(THF), 3 equivalents of lithium aluminum hydride (LAH) was slowly addedat 0° C. and stirred for 12 hours. The reaction was quenched with asaturated aqueous solution of 10% NaOH and H₂O and extracted with ether.The ether layer was dried with Na₂SO₄ and filtered, and the solvent wasremoved by distillation under reduced pressure. After purification bycolumn chromatography. 8a-c was obtained in yields of 22.5% and 88.7%.

Compounds 14a to 14i were synthesized in the same manner as in ReactionScheme 5 below.

entry R compound 1

14a 2

14b 3

14c 4

14d 5

14e 6

14f 7

14g 8

14h 9

14i

3. Methyl 2,4-dihydroxybenzoate (9)

2,4-dihydroxybenzoic acid (10.00 g, 64.88 mmol) and sulfuric acid(H₂SO₄) of 5 mL were added to 40 mL of MeOH and refluxed at 100° C. for24 hours. After cooling at room temperature, the solvent was removed bydistillation under reduced pressure, and H₂O was added at 0° C. Theresulting white solid was dissolved in EA and washed with a saturatedaqueous NaHCO₃ solution. The EA layer was dried with Na₂SO₄ andfiltered, and the solvent was removed by distillation under reducedpressure to obtain Compound 9 in a yield of 85%.

¹H NMR (400 MHz, CDCl₃) d 11.0 (s, 1H), 7.73 (d, J=8.4 Hz, 1H),6.41-6.37 (m, 2H), 5.78 (s, 1H), 3.92 (s, 3H).

4. Methyl 5-chloro-2,4-dihydroxybenzoate (10)

Compound 8 (8.30 g, 49.41 mmol) and sulfuryl chloride (SO₂Cl₂) (4.11 mL,56.83 mmol) were added to DCM and stirred at room temperature for 24hours. The solvent was removed by distillation under reduced pressure,dissolved in EA, washed with a saturated aqueous NaHCO₃ solution, driedover Na₂SO₄ and filtered, and the solvent was removed by distillationunder reduced pressure. It was purified by column chromatography using asolvent mixture of EA:hexane=1:9 to obtain Compound 10 in a yield of32.5%.

¹H NMR (400 MHz, CDCl₃) d 10.8 (s, 1H), 7.82 (s, 1H), 6.61 (s, 1H), 5.91

5. Methyl 2,4-bis (allyloxy)-5-chlorobenzoate (11)

Compound 9 (1.10 g, 5.46 mmol), allyl bromide (1.23 mL, 14.20 mmol) andK₂CO₃ (1.96 g, 14.20 mmol) were dissolved in DMF. It was stirred underargon at room temp me for 12 hours, then dissolved in EA and washed witha saturated aqueous NaHCO₃ solution. The EA layer was dried over Na₂SO₄,and the solvent was removed by distillation under reduced pressure toobtain Compound 11 in a yield of 83%.

¹H NMR (400 MHz, CDCl₃) d 7.85 (s, 1H), 7.25 (s, 1H), 6.44-5.95 (m, 2H),5.50-5.40 (m, 2H), 5.30-5.26 (m, 2H), 4.58-4.45 (m, 4H), 3.81 (s, 3H).

6. 2,4-bis(allyloxy)-5-chlorobenzoic Acid (12)

Compound 8 (1.27 g, 5.64 mmol) and NaOH (1.12 g, 28.1 mmol) were addedto H₂O (20 mL) and MeOH (20 mL) and stirred at room temperature for 8hours, and then was dissolved in EA and washed with 3N HCl. The EA layerwas dried over Na₂SO₄ and then the solvent was removed by distillationunder reduced pressure to obtain Compound 12 in a yield of 74%.

¹H NMR (400 MHz, CDCl₃) d 8.16 (s, 1H), 6.54 (s, 1H), 6.10-5.99 (m, 2H),5.51-5.35 (m, 2H), 4.76-4.65 (m, 4H).

7. N-Benzyl-5-chloro-2,4-dihydroxybenzamide (14a)

Compound 12 (0.50 g, L86 mmol), benzylamine (0.26 g, 1.86 mmol), HOBt(0.25 g, 1.86 mmol), DCC (0.77 g, 3.72 mmol) and DIPEA (0.32 mL, 1.86mmol) were dissolved in DMF and microwave reaction was performed at 80°C. and 20 bar for 3 hours. DMF was removed by distillation under reducedpressure and Compound 13a was obtained by purifying by MPLC (R_(f)=0.19,EA:hexane 1:4). Compound 13a vas dissolved in THF, and PdCl₂(PPh₃)₂ (20mg) and NH₄HCO₂ (200 mg) were added thereto, followed by microwavereaction at 120° C. and 20 bar for 30 minutes.

It was dissolved in EA, washed with H₂O, and the EA layer was dried withNa₂SO₄ followed by filtering. The solvent was removed by distillationunder reduced pressure, and Compound 14a was obtained purifying by MPLC(R_(f)=0.13, EA:hexane=3:7) in yield of 53.2%.

¹H NMR (400 MHz, MeOD) d 7.77 (s, 1H), 7.29-7.26 (m, 4H), 7.22-7.19 (m,1H), 6.40 (s, 1H), 4.50 (s, 2H). ¹³C NMR (100 MHz, MeOD) d 165.2, 157.0,154.2, 135.3, 125.2, 124.7, 123.7, 123.4, 108.1, 104.9, 100.2, 39.1.

8. N-Benzyl-5-chloro-2,4-dihydroxy-n-methylbenzamide (14b)

Compound 12 (0.30 g, 1.12 mmol), Compound 6a (0.21 g, 1.67 mmol), HOBt(0.15 g, 1.12 mmol), DCC (0.46 g, 2.23 mmol) and DIPEA (0.19 mL, 1.12mmol) were dissolved in DMF and microwave reaction was performed at 80°C. and 20 bar for 3 hours. It was then dissolved in EA and washed withH₂O, and the EA layer was dried with NaSO₄ and filtered, and then thesolvent was removed by distillation under reduced pressure, it waspurified by MPLC (R_(f)=0.31, EA:hexane=3:7) to obtain Compound 13b.Compound 13b was dissolved in THF, and PdCl₂(PPh₃)₂ (20 mg) and NH₄HCO₂(200 mg) were added thereto, followed by microwave reaction at 120° C.and 20 bar for 30 minutes. After dissolving in EA and washing with H₂O,the EA layer was dried with Na₂SO₄ and filtered, and the solvent wasremoved by distillation under reduced pressure. Compound 14b wasobtained by purifying by MPLC (R_(f)=0.28, EA:hexane=2:3) at a yield of62.5%.

¹H NMR (400 MHz, MeOD) d 7.35-7.24 (m, 5H), 7.16 (s, 1H), 6.50 (s, 1H),2.89 (s, 3H).

9. 5-chloro-2,4-dihydroxy-n-(4-methoxybenzyl)-n-methylbenzamide (14c)

Compound 12 (0.30 g, 1.12 mmol), Compound 6b (0.18 g, 1.67 mmol), HOBt(0.15 g, 1.12 mmol), DCC (0.46 g, 2.23 mmol) and DIPEA (0.19 mL, 1.12mmol) were dissolved in DMF and microwave reaction was performed at 80°C. and 20 bar for 3 hours. DMF was removed by distillation under reducedpressure, and Compound was obtained by purifying by MPLC (R_(f)=0.20,EA:hexane=3:7). Compound 13c was dissolved in THF, and PdCl₂(PPh₃)₂ (20mg) and NH₄HCO₂ (200 mg) were added thereto, followed by microwavereaction at 120° C. and 20 bar for 30 minutes. After dissolving in EAand washing with H₂O, the EA layer was dried with Na₂SO₄ and filtered,and the solvent was removed by distillation under reduced pressure.

Compound 14c was obtained by purifying by MPLC (R_(f)=0.30, EA:hexane=2:3) in a yield of 63.8%.

¹H NMR (400 MHz, CDCl₃) d 7.27 (s, 1H), 7.20 (d, J=8.4 Hz, 2H), 6.90 (d,J=6.8 Hz, 2H), 6.62 (s, 1H), 4.64 (s, 2H), 3.80 (s, 3H), 3.03 (s, 3H).¹³C NMR (100 MHz, CDCl₃) d 171.1, 159.6, 159.3, 155.2, 129.0, 128.8,128.0, 114.4, 111.1, 110.5, 105.2, 55.4.

10. 5-chloro-n-(3,4-dimethoxybenzyl)-2,4-dihydroxy-n-methylbenzamide(14d)

Compound 12 (0.33 g, 1.23 mmol) Compound 6c (0.33 g, 1.84 mmol), HOBt(0.17 g, 1.23 mmol), DCC (0.51 g, 2.46 mmol) and DIPEA (0.22 mL, 1.23mmol) were dissolved in DMF and microwave reaction was performed at 120°C. and 20 bar for 3 hours. Compound 13d was obtained by purifying byMPLC (R_(f)=0.26, EA:hexane=2:3). Compound 13d was dissolved in THF, andPdCl₂(PPh₃)₂ (20 mg) and NH₄HCO₂ (200 mg) were added thereto, followedby microwave reaction at 120° C. and 20 bar for 30 minutes. Afterdissolving in EA and washing with H₂O, the EA layer was dried withNa₂SO₄ and filtered, and the solvent was removed by distillation underreduced pressure. Compound 14d was obtained by purifying by MPLC(R_(f)=0.22, EA:hexane=1:1) in a yield of 63.8%.

¹H NMR (400 MHz, CDCl₃) d 7.31 (s, 1H), 6.88-6.81 (m, 3H), 6.66 (s, 1H),4.65 (s, 2H), 3.88 (d, J=6.4 Hz, 6H), 3.07 (s, 3H). ¹³C NMR (100 MHz,CDCl₃) d 170.5, 155.3, 149.1, 148.3, 131.9, 131.8, 128.8, 128.7, 128.6,128.5, 119.3, 111.0, 110.5, 105.1, 55.7.

11.N-(benzo[d][1,3]dioxol-5-ylmethyl)-5-chloro-2,4-dihydroxy-n-methylbenzamide(14e)

Compound 12 (0.30 g, 1.12 mmol), Compound 6d (0.28 g, 1.67 mmol), HOBt(0.15 g, 1.12 mmol), DCC (0.46 g, 2.23 mmol) and DIPEA (0.19 mL, 1.12mmol) were in DMF and microwave reaction vas performed at 80° C. and 20bar for 3 hours. DMF was removed by distillation under reduced pressureand then Compound 13e was obtained by purifying by MPLC (R_(f)=0.25,EA:hexane=3:7). Compound 13e was dissolved in Tiff, and PdCl₂(PPh₃)₂ (20mg) and NH₄HCO₂ (200 mg) were added thereto, and microwave reaction wasperformed at 120° C. and 20 bar for 30 minutes. After dissolving in EAand washing with H₂O, the EA layer was dried with Na₂SO₄ and filtered,and the solvent was removed by distillation under reduced pressure andCompound 14e was obtained by purifying by MPLC (R_(f)=0.23,EA:hexane=2.3) in a yield of 78.6%.

¹H NMR (400 MHz, CDCl₃) d 7.28 (s, 1H), 6.79-6.72 (m, 3H), 6.60 (s, 1H),5.96 (s, 2H), 4.59 (s, 2H), 3.03 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) d

12. 5-chloro-2,4-dihydroxy-n-methyl-n-(3,4,5-trimethoxybenzyl) benzamide(14f)

Compound 1.2 (0.30 g, 1.12 mmol), Compound 6e (0.35 g, 1.23 mmol), (0.15g, 1.12 mmol), DCC (0.46 g, 2.23 mmol) and DIPEA (0.19 mL, 1.12 mmol)were dissolved in DMF and microwave reaction was performed at 80° C. and20 bar for 3 hours. Compound 13f was obtained by purifying by MPLC(R_(f)=0.20, EA:hexane=2:3). Compound 13f vas dissolved in THF andPdCl₂(PPh₃)₂ (20 mg) and NH₄HCO₂ (200 mg) were added thereto, followedby microwave reaction at 120° C. and 20 bar for 30 minutes. Afterdissolving in EA and washing with H₂O, the EA layer was dried withNa₂SO₄ and filtered, and the solvent was removed by distillation underreduced pressure and Compound 14f was obtained by purifying by MPLC(R_(f)=0.26, EA:hexane=3:2) in yield of 67.0%.

¹H NMR (400 MHz, CDCl₃) d 7.23 (s, 1H), 6.56 (s, 1H), 6.47 (s, 1H), 4.59(s, 2H), 3.80 (d, J=1.2 Hz, 9H), 3.02 (s, 3H). ¹³C NMR (100 MHz, CDCl₃)d 171.2, 158.7, 155.4, 153.6, 137.3, 132.0, 128.8, 111.6, 110.8, 105.1,104.5, 61.0, 56.2.

13.5-chloro-n-(2-chloro-3,4,5-trimethoxybenzyl)-2,4-dihydroxy-n-methylbenzamide(14 g)

Compound 12 (0.20 g, 0.74 mmol), Compound 6f (0.27 g, 1.12 mmol), HOBt(0.10 g, 0.74 mmol), DCC (0.31 g, 1.49 mmol) and DIPEA (0.15 mL, 0.74mmol) were dissolved in DMF and microwave reaction was performed at 120°C. and 20 bar for 3 hours. After washing with EA and washing with H₂O,the EA layer was dried with NaSO₄, filtered, and the solvent was removedby distillation under reduced pressure and Compound 13g was obtained bypurifying by MPLC (R_(f)=0.26, EA:hexane=3:7). Compound 13g wasdissolved in THF, PdCl₂(PPh₃)₂ (20 mg) and NH₄HCO₂ (200 mg) were addedand microwave reaction was performed at 120° C. and 20 bar for 30minutes, was dried over Na₂SO₄ and filtered, and the solvent was removedby distillation under reduced pressure. Compound 14g was obtained bypurifying by MPLC (R_(f)=0.20, EA:hexane=1:1) in a yield of 41.9%.

¹H NMR (400 MHz, CDCl₃) d 7.21 (s, 1H), 6.75 (s, 1H), 6.47 (s, 1H), 4.75(s 2H), 3.90 (d, J=9.2 Hz, 6H), 3.83 (s, 3H), 3.03 (s, 3H). ¹³C NMR (100MHz, CDCl₃) d 171.1, 156.3, 155.4, 152.5, 150.0, 142.4, 132.0, 129.3,128.9, 119.2, 113.0, 111.3, 107.0, 104.2, 61.2, 56.1, 49.8.

14.N-(2-bromo-3,4,5-trimethoxybenzyl)-5-chloro-2,4-dihydroxy-n-methylbenzamide(14h)

Compound 12 (019 g, 0.69 mmol), Compound 6 g (0.30 g, 1.03 mmol), HOBt(0.09 g, 0.69 mmol) DCC (028 g. 1.38 mmol) and DIPEA (0.12 mL, 0.69mmol) was dissolved in DMF and microwave reaction was performed at 120°C. and 20 bar for 3 hours. After dissolving in EA and washing with H₂O,the EA layer was dried with Na₂SO₄ and filtered, and the solvent wasremoved by distillation under reduced pressure. Compound 13h wasobtained by purifying by MPLC (R_(f)=0.24, EA:hexane 3:7).

Compound 13h was dissolved in THF, and PdCl₂(PdCl₂(PPh₃)₂ (20 mg) andNH₄HCO₂ (200 mg) were added thereto, followed by microwave reaction at120° C. and 20 bar for 30 minutes. After dissolving in EA and washingwith H₂O, the EA layer was dried with Na₂SO₄ and filtered, and thesolvent was removed by distillation under reduced pressure. Compound 14hwas obtained by purifying by MPLC (R_(f)=24, EA:hexane=1:1) in yield of20.5%.

¹H NMR (400 MHz, CDCl₃) d 7.15 (s, 1H), 6.69 (s, 1H), 6.42 (s, 1H), 4.68(s, 2H), 3.83 (d, J=6.0 Hz, 6H), 3.77 (d, 3H), 2.98 (s, 3H). ¹³C NMR(100 MHz, CDCl₃) d 160.8, 155.2, 153.4, 151.4, 143.0, 130.9, 128.6,110.7, 110.3, 105.4, 61.4, 61.3, 60.7, 56.5.

15. 5-chloro-2,4-dihydroxy-n-methyl-n-(3-methylbenzyl)benzamide (14i)

Compound 12 (0.25 g, 0.93 mmol), Compound 6h (0.19 g, 1.40 mmol), HOBt(0.13 g, 0.93 mmol), DCC (0.39 g, 1.86 mmol) and DIPEA (0.17 mL, 0.93mmol) were in DMF and microwave reaction was performed at 120° C. and 20bar for 3 hours. After dissolving in EA and washing with H₂O, the EAlayer was dried with Na₂SO₄ and filtered, and the solvent was removed bydistillation under reduced pressure and Compound 13i was obtained bypurifying by MPLC (R_(f)=0.22, EA:hexane=1:4).

Compound 13i was dissolved in THF, and PdCl₂(PPb₃)₂ (20 mg) and NH₄HCO₂(200 mg) were added thereto, and microwave reaction was performed at120° C. and 20 bar for 30 minutes. After dissolving in EA and washingwith H₂O, the EA layer was dried with Na₂SO₄ and filtered, and thesolvent was removed by distillation under reduced pressure and Compound14i was obtained by purifying by MPLC (R_(f)=0.20, EA:hexane=3:7) in ayield of 68.7%.

¹H NMR (400 MHz, CDCl₃) d 7.31 (s, 1H), 7.28 (d, J=7.6 Hz, 1H), 7.15 (d,J=7.6 Hz, 1H), 7.10-7.09 (m, 2H), 6.65 (s, 1H), 4.70 (s, 2H), 3.08 (s,3H), 2.38 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) d 171.3, 160.0, 155.2,138.8, 136.0, 128.9, 128.8, 128.7, 128.3, 124.6, 110.9, 110.4, 105.2,36.8.

Compounds 16a and 16b were synthesized in the same manner as in ReactionScheme 6 below.

entry R compound 10

16a 11

16b

16. 5-chloro-2,4-dihydroxybenzoic Acid (15)

Compound 9 (1.98 g, 9.77 mmol) and NaOH (1.95 g, 48.80 mmol) were addedto H₂O (30 mL) and MeOH (30 mL) and stirred at room temperature for 12hours. It was dissolved in EA, washed with 3N HCl saturated aqueoussolution, and the EA layer was dried with Na₂SO₄ and filtered. Thesolvent was removed by distillation under reduced pressure to obtainCompound 15 in a yield of 100% by purifying by MPLC.

¹H NMR (400 MHz, CDCl₃) d 7.76 (s, 1H), 6.41 (s, 1H).

17. 5-chloro-2,4-dihydroxy-n-methyl-n-(2-methylbenzyl)benzamide (16a)

Compound 15 (0.17 g, 0.92 mmol), Compound 8a (0.19 g, 1.40 mmol), HOBt(0.12 g, 0.92 mmol), EDC (0.35 g, 1.86 mmol) and DIPEA (0.16 mL, 0.92mmol) were dissolved in DMF and microwave reaction was performed at 120°C. and 20 bar for 3 hours. It was dissolved in EA, washed with asaturated aqueous solution of 1N HCl, dried over Na₂SO₄ and filtered.The solvent was removed by distillation under reduced pressure to obtainCompound 16a in a. The residue was purified by MPLC (R_(f)=0.23,EA:hexane=1:4) to obtain Compound 16a in a yield of 90.5%.

¹H NMR (400 MHz, CDCl₃) d 7.25-7.24 (m, 4H), 6.65 (s, 1H), 4.72 (s, 2H),3.07 (s, 3H), 2.27 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) d 160.9, 155.2,136.4, 134.0, 131.0, 128.7, 128.0, 127.4, 126.7, 110.7, 110.2, 105.3,19.2.

18. 5-chloro-2,4-dihydroxy-n-methyl-n-(4-methylbenzyl)benzamide (16b)

Compound 15 (0.21 g, 1.12 mmol), Compound 8b (0.23 g, 1.69 mmol), HOBt(0.15 g, 1.12 mmol), EDC (0.43 g, 2.25 mmol) and DIPEA (0.20 mL, 1.12mmol) were dissolved in DMF Microwave reaction was performed at 120° C.and 20 bar for 3 hours. It was dissolved in EA, washed with 3N HClsaturated aqueous solution, and then the EA layer was dried with Na₂SO₄,filtered. The solvent was removed by distillation under reduced pressureto obtain Compound 16b in a yield of 26.0% by purifying by MPLC(R_(f)=0.23, EA:hexane=1:4).

¹H NMR (400 MHz, CDCl₃) d 7.28 (s, 1H), 7.17 (t, J=9.2 Hz, 9.2 Hz, 4H),6.63 (s, 1H), 4.67 (s, 2H), 3.04 (s, 3H), 2.35 (s, 3H). ¹³C NMR (100MHz, CDCl₃) d 160.1, 155.0, 137.7, 133.0, 129.7, 128.6, 128.5, 127.6,127.3, 110.9, 110.2, 105.2, 21.2.

Compounds 21a to 21f were synthesized in the same manner as in ReactionScheme 7 below.

19. Methyl 2,4-dihydroxy-5-isopropylbenzoate (17)

Compound 9 (10.70 g, 63.50 mmol), AlCl₃ (16.90 g, 127.0 mmol) and2-bromopropane (11.90 mL, 127.0 mmol) were dissolved in DCM (125 mL) andrefluxed at 50° C. for 24 hours under argon injection. 2 equivalents ofAlCl₃ and 2-bromopropane were added, respectively in a 6-hour period.The reaction mixture was adjusted to pH 6 with a saturated aqueoussolution of 10% NaOH and the solvent was removed by distillation underreduced pressure, and the residue was dissolved in EA and washed with asaturated aqueous NaHCO₃ solution. The EA layer was dried with Na₂SO₄,filtered and the solvent was removed by distillation under reducedpressure. Compound 17 was obtained by purifying by column chromatographyusing a solvent mixed in EA:hexane=1:9, in a yield of 34.5%.

¹H NMR (400 MHz, CDCl₃) d 12.61 (s, 1H), 7.52 (s, 1H), 6.35 (s, 1H),6.31 (s, 1H), 3.22-3.12 (m, 1H), 2.61 (s, 3H), 1.27 (d, J=6.8 Hz, 6H).¹³C NMR (100 MHz, CDCl₃) d 170.7, 161.6, 159.6, 128.1, 127.1, 105.7,103.2, 52.2, 26.7, 22.8.

20. 2,4-dihydroxy-5-isopropylbenzoic Acid (18)

Compound 17 (4.60 g, 21.90 mmol) and LiOH (10 g) were added into H₂O (30mL) and MeOH (30 mL) at 70° C. and refluxed for 12 hours. It wasdissolved in EA and washed with 3N HCl saturated aqueous solution. TheEA layer was dried with Na₂SO₄, filtered and the solvent was removed bydistillation under educed pressure to obtain Compound 18 in a yield of60.5%.

¹H NMR (400 MHz, CDCl₃) d 7.68 (s, 1H), 6.30 (s, 1H), 3.19-3.12 (m, 1H),1.17 (d, J=4.0 Hz, 6H).

21. Methyl 4-((5-chloro-2,4-dihydroxy-n-methylbenzamido)methyl) benzoate(19a) and Methyl4-((2,4-Dihydroxy-5-Isopropyl-N-Methylbenzamido)Methyl)Benzoate (19b)

1 equivalent of Compound 12 or 18, 1.5 equivalents of Compound 8c, 1equivalent of HOBt, 2 equivalents of EDC and 1 equivalent of DIPEA weredissolved in DMF and microwave reaction was performed at 120° C. and 20bar for 3 hours. After dissolving in EA and washing with H₂O, the EAlayer was dried with NaSO₄, filtered, and then the solvent was removedby distillation under reduced pressure. Compound 19a or 19b was obtainedby purifying by MPLC according to the conditions of each compound inyields of 76.1%, 81.7%, respectively.

19a: ¹H NMR (500 MHz, CDCl₃) d 8.03 (d, J=6.6 Hz, 2H), 7.35 (d, J=6.6Hz, 2H), 7.25 (s, 1H), 6.62 (s, 1H), 4.75 (s, 2H), 3.91 (s, 3H), 3.07(s, 3H).

19b: ¹H NMR (500 MHz, CDCl₃) d 8.05 (d, J=6.7 Hz, 2H), 7.37 (d, J=6.7Hz, 2H), 7.11 (s, 1H), 6.42 (s, 1H), 4.76 (s, 2H), 3.92 (s, 3H),3.13-3.04 (m, 1H), 3.05 (s, 3H), 0.94 (d, J=5.3 Hz, 6H).

22. 4-((5-chloro-2,4-dihydroxy-n-methylbenzamido)methyl)benzoic Acid(20a) and 4-((2,4-dihydroxy-5-isopropyl-n-methylbenzamido)methyl)benzoicAcid (20b)

1 equivalent of Compound 19a or 19b and LiOH (2.0 g) were added to H₂O(20 mL) and MeOH (20 mL) and stirred at room temperature for 3 hours. Itwas dissolved in EA and washed with 3N HCl saturated aqueous solution.The EA layer was dried with Na₂SO₄ and filtered, and the solvent wasremoved by distillation under reduced pressure to obtain Compounds 20aand 20b at a yield of 84.8% and 88.1%, respectively.

20a: ¹H NMR (500 MHz, CDCl₃) d 8.13 (d, J=6.66 Hz, 2H), 7.41 (d, J=6.6Hz, 2H), 7.30 (s, 1H), 6.68 (s, 1H), 4.79 (s, 2H), 3.12 (s, 3H).

20b: ¹H NMR (500 MHz, MeOD) d 8.00 (d, J=6.5 Hz, 2H), 7.41 (d, J=5.8 Hz,2H), 7.0 (s, 1H), 6.34 (s, 1H), 4.86 (s, 2H), 3.18-3.11 (m, 1H), 3.0 (s,3H), 1.11 (d, J=5.2 Hz, 6H).

23. 5-chloro-2,4-dihydroxy-n-methyl-n-(4-(methylcarbamoyl)benzyl)benzamide (21a)

Compound 20a (0.27 g, 0.79 mmol), 40% methylamine in d-water (0.10 mL,1.18 mmol), HOBt (0.11 g, 0.79 mmol), EDC (0.30 g, 1.58 mmol) and DIPEA(0.14 mL, 0.79 mmol) was dissolved in DMF and microwave reaction wasperformed at 120° C. and 20 bar for 2 hours. It was dissolved in EA,washed with a saturated aqueous solution of 1N HCl, dried over Na₂SO₄and filtered, and the solvent was removed by distillation under reducedpressure. Compound 21a was obtained by purifying by MPLC (C18 column,R_(f)=0.21, H₂O:MeOH=4:1) in a yield of 16.7%.

¹H NMR (500 MHz, CDCl₃) d 7.78 (d, J=6.6 Hz, 2H), 7.36 (d, J=6.6 Hz,2H), 7.30 (s, 1H), 6.68 (s, 1H), 4.75 (s, 2H), 3.09 (s, 3H), 3.03 (d,J=3.8 Hz, 3H).

24.5-chloro-n-(4-(ethylcarbamoyl)benzyl)-2,4-dihydroxy-n-methylbenzamide(21b)

Compound 20a (0.27 g, 0.79 mmol), 70% ethylamine in H₂O (0.10 mL, 1.18mmol), HOBt (0.11 g, 0.79 mmol), EDC (0.30 g, 1.58 mmol) mL, 0.79 mmol)were dissolved in DMF and microwave reaction was performed at 120° C.and 20 bar for 2 hours. After dissolving in EA and washing with IN HClsaturated aqueous solution, the EA layer is dried with Na₂SO₄ andfiltered, and the solvent is removed by distillation under reducedpressure. Compound 21b was obtained by purifying by MPLC (C18 column,R_(f)=0.21, H₂O:MeOH=4:1) in a yield of 31.2%.

¹H NMR (500 MHz, CDCl₃) d 7.78 (d, J=6.6 Hz, 2H), 7.35 (d, J=6.6 Hz,2H), 7.30 (s, 1H), 6.67 (s, 1H), 4.75 (s, 2H), 3.54-3.49 (m, 2H), 2.96(s, 3H), 1.26 (t, J=5.8 Hz, 5.8 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) d

25.5-chloro-2,4-dihydroxy-n-methyl-n-(4-(propylcarbamoyl)benzyl)benzamide(21c)

Compound 20a (0.26 g, 0.76 mmol), propylamine (0.10 mL, 1.14 mmol), HOBt(0.10 g, 0.76 mmol), EDC (0.30 g, 1.52 mmol) and DIPEA (0.10 mL, 0.76mmol) were dissolved in DMF and microwave reaction was performed at 120°C. and 20 bar for 3 hours. After dissolving in EA and washing with 1NHCl saturated aqueous solution, the EA layer was dried with NaSO₄,filtered and the solvent was removed by distillation under reducedpressure. Compound 21c was obtained by purifying by MPLC (C18 column,R_(f)=0.21, H₂O:MeOH=7:3) in a yield of 45.5%.

¹H NMR (500 MHz, CDCl₃) d 7.78 (d, J=6.6 Hz, 2H), 7.35 (d, J=6.6 Hz,2H), 7.28 (s, 1H), 6.67 (s, 1H), 4.75 (s, 2H), 3.46-3.42 (m, 2H), 3.09(s, 3H), 1.67-1.62 (m, 2H), 1.00 (t, J=5.9 Hz, 5.9 Hz, H).

26. 2,4-dihydroxy-5-isopropyl-n-methyl-n-(4-(methylcarbamoyl)benzyl)benzamide (21d)

Compound 20b (0.21 g, 0.62 mmol), 40% methylamine in d-water (0.08 mL,0.93 mmol), HOBt (0.08 g, 0.62 mmol), EDC (0.23 g, 1.24 mmol) and DIPEA(0.11 mL, 0.62 mmol) were dissolved in DMF, and microwave reaction wasperformed at 120° C. and 20 bar for 3 hours. This was dissolved in EA,washed with a saturated aqueous solution of 1N HCl, dried over Na₂SO₄and filtered, and the solvent was removed by distillation under reducedpressure. Compound 21d was obtained by purifying by MPLC (C18 column,R_(f)=0.21, H₂O:MeOH=7:3) in a yield of 36.1%.

¹H NMR (500 MHz, CDCl₃) d 7.79 (d, J=6.5 Hz, 2H), 7.36 (d, J=6.4 Hz,2H), 7.08 (s, 1H), 6.41 (s, 1H), 6.46 (s, 2H), 3.08 (s, 3H), 3.06-3.03(m, 1H), 0.98 (d, J=5.3 Hz, 6H).

27.N-(4-(ethylcarbamoyl)benzyl)-2,4-dihydroxy-5-isopropyl-n-methylbenzamide(21e)

Compound 20b (0.22 g, 0.64 mmol), 70% ethylamine in d-water (0.08 ME,0.97 mmol), HOBt (0.09 g, 0.64 m EDC (025 g, 1.29 mmol) and DIPEA (0.12mL, 0.64 mmol) were dissolved in DMF, and microwave reaction wasperformed at 120° C. and 20 bar for 3 hours. It was dissolved in EA,washed with a saturated aqueous solution of 1N HCl, dried over Na₂SO₄and filtered, and the solvent was removed by distillation under reducedpressure. Compound 21e was obtained by purifying by MPLC (C18 column,R_(f)=0.21, H₂O:MeOH=7:3) in a yield of 24.3%.

¹H NMR (500 MHz, CDCl₃) d 7.78 (d, J=6.6 Hz, 2H), 7.33 (d, J=6.6 Hz,2H), 7.05 (s, 1H), 6.43 (s, 1H), 4.73 (s, 2H), 3.52-3.47 (m, 2H),3.07-3.05 (m, 1H), 3.05 (s, 3H), 1.27-1.23 (m, 3H), 0.98 (d, J=5.4 Hz,6H).

28. 2,4-dihydroxy-5-isopropyl-n-methyl-n-(4-(propylcarbamoyl)benzyl)benzamide (21f)

Compound 20b (0.22 g, 0.64 mmol), propylamine (0.08 mL, 0.96 mmol), HOBt(0.09 g, 0.64 mmol), EDC (0.25 g, 1.28 mmol)), DIPEA (0.11 mL, 0.64mmol) were dissolved in DMF and microwave reaction was performed at 120°C. and 20 bar for 3 hours. It was dissolved in EA, washed with asaturated aqueous solution of 1N HCl, dried over Na₂SO₄, filtered, andthen the solvent was removed by distillation under reduced pressure.Compound 21f was obtained by purifying by MPLC (C18 column, R_(f)=0.21,H₂O:MeOH=7:3) in a yield of 22.3%.

¹H NMR (500 MHz, CDCl₃) d 7.76 (d, J=6.6 Hz, 2H), 7.29 (d, J=6.6 Hz,2H), 7.02 (s, 1H), 6.43 (s, 1H), 4.70 (s, 2H), 3.41-3.37 (m, 2H),3.07-3.05 (m, 1H), 3.02 (s, 3H), 1.63 (s, 2H), 0.97-0.92 (m, 9H).

29. N-benzyl-2,4-dihydroxy-5-isopropyl-n-methylbenzamide (21 g)

2,4-Dihydroxy-5-isopropylbenzoic acid (0.33 g, 1.70 mmol),N-methylaniline (0.33 g, 2.54 mmol), EDC (0.65 g, 3.30 mmol) and DIPEA(0.30 mL, 1.70 mmol) were dissolved in DMF and microwave reaction wasperformed at 120° C. and 20 bar for 3 hours. It was dissolved in EA,washed with a saturated aqueous solution of 1N HCl, dried with Na₂SO₄,filtered, and then the solvent was removed by distillation under reducedpressure. After purification using a mixed solvent of EA:hexane=1:4(R_(f)=0.18), Compound 21g was obtained in a yield of 15.5%.

¹H NMR (500 MHz, CDCl₃)δ10.5(s,1H), 7.42-7.39(m,2H), 7.34-7.29(m,3H),6.38(s,1H), 5.34(s,1H), 4.74(s,2H), 3.09(s,3H), 3.06-2.95(m,1H), 0.96(d,J=5.4 Hz, 6H).

EXAMPLE 4 Evaluation of Biological Activity of Benzamide Compounds

1. Evaluation of Binding Ability to HSP90

Fluorescence polarization assay was performed to confirm the bindingability to the compound synthesized with HSP90 according theconcentration. 1 M dithiothreitol (DTT), 10 mg/m: BGG (bovine gammaglobulin) were added to hexafluorobenzene (HFB) buffer (20 mM HEPES pH7.3, 50 mM KCl, 5 mM MgCl₂, 20 mM Na₂MoO₄, 0.01% NP₄O, 3^(rd) distilledwater) and mixed. Then, 100 nM gambogic acid (GA)-FITC (Fluoresceinisothiocyanate) was added and reacted at room temperature for 10minutes. Each 100 μL of the mixture was dispensed into the control wellsof a 96-well plate, 2 μL of HSP90α was added to the remaining mixture,and each 100 μL of the other wells was dispensed. Each 98 μL wasdispensed into the wells to which the compound was to be treated, andthen each 2 μL was added at 5 mM, 2.5 mM, 500 μM, 50 μM, 25 μM, 5 μM,500 nM or 50 nM for each concentration. After 1, 4, 6, or 18 hours ofreaction, fluorescence polarization was measured at 495/530 nm by aplate reader. The binding ability of the compound to HSP90 was expressedas mP (millipolarization unit=1000 mP unit). The results are shown inTable 3 below.

2. Measurement of Cell Viability

To confirm the cell viability according to the concentration of thecompound synthesized in H1975 cells (ATCC) resistant to Gefitinib, MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-methylpiperidin-3-y)-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-terazolium,inner salt] analysis was performed. When the cells were cultured so asto occupy 80% of the bottom of the culture dish, the cells were detachedand diluted with RPMI 1640 medium to a concentration of 1.5×10³cells/well. Each 100 μL of the suspension was added to each well of a96-well plate and cultured for 14 hours. Then, the medium was changed tothe culture solution treated with compound of each concentration eachconcentration (1a-g: 0, 1, 5, 10, 30, 50 or 100 μM, 14a-I, 16a, b,21a-f: 0, 0.01, 0.1, 1, 5, 10, 30, 50, 70 or 100 μM, and cultured for 3days. Each 20 μL of the NITS solution was dispensed into each well andreacted for 1 hour at 37° C. in an incubator supplied with 5% CO₂. Theabsorbance was measured at 490/690 nm for 1 hour using a microplatereader, and the cell viability according to concentration was calculatedas a percentage (%) and an IC₅₀ (μM). The results are shown in Table 3below

TABLE 3 HSP90 H1975 FP) (IC₅₀; (EC₅₀; compound R₁ R₂ nM) μM) 14a Cl

6683 86.2 14b Cl

413 35.5 14c Cl

135 16.7 14d Cl

362 33.4 14e Cl

585 103 14f Cl

1461 73.1 14g Cl

429 33.8 14h Cl

528 34.3 14i Cl

995 47.5 16a Cl

830 49.3 16b Cl

558 34.3 21a Cl

496.0 34.8 21b Cl

611.5 21.7 21c Cl

454.0 20.8 21d isopropyl

11.4 0.83 21e Isopropyl

6.5 0.46 21f isopropyl

5.3 0.42 21g isopropyl

33 4.1

As a result of measuring the binding ability between the benzamidecompounds 14a to 14i, 16a, 16b and 21a to 21f and HSP90 according to thepresent invention shown in the Table 3, the remaining compounds otherthan 14a and 14f exhibited a high binding ability at the nanomol levelconcentration. Among them, Compound 21f showed the best binding abilitywith IC₅₀ of 5.3 nM.

Furthermore, as a result of the cell viability of the compound wasmeasured in H1975 cells shown in the Table 3, it was confirmed that cellproliferation was inhibited by all benzamide compounds in atime-dependent manner. In particular, Compound 21f exhibited goodefficacy with an EC₅₀ of 420 nM.

3. Analysis of Protein Expression

Compound 21f having good efficacy was selected and HSP90 client proteinproliferation effect in the same cell line was confirmed, in which theHSP90 client proteins were Her2 (human EGFR-related 2), EGFR (epidermalgrowth factor receptor), Met and c-Raf were used.

H1975 cells resistant to Gefitinib were cultured in a mediumsupplemented with 10% fetal bovine serum (FBS) for 24 hours, and thecells were detached, harvested and diluted with a RPMI 1640 medium so asto be 5×10⁵ cells/well. Each 5 mL of the suspension was added to 6 mmculture dish and cultured for 24 hours. Then, the medium was changed tothe culture solution treated with each compound of concentration (21f 0,0.05, 0.1, 0.5 or 1 μM, GA: 1 μM), and cultured for 24 hours. Afterculture, the cells were harvested and centrifuged at 1300 rpm for 5minutes at 4° C., and the supernatant was removed. The pellet was washedwith phosphate buffered saline, centrifuged once again under the sameconditions, and the supernatant was removed. Lysis buffer was added tothe remaining pellet and lysed by vortexing at 5 minute intervals for 30minutes. The supernatant was separated by centrifugation at 16000 rcffor 4 minutes at 4° C., and proteins were quantified using BCA (bovineserum albumin) kit. SDS-PAGE (sodium dodecylsulfate-polyacrylamide gelelectrophoresis) was performed using 10, 12% running gel and 4% stackinggel (10 mA per gel). The separated proteins were transferred to animmunoblot PVDF, membrane (polyvinylidene fluoride membrane) at 100 Vfor 75 minutes. The membranes were blocked with 5% skim milk solutionfor 2 hours and then added a primary antibody diluted in the ratio of1:750-1,000 in 0.1% tween-20 solution and reacted at 4° C. for 12 hours.After washing three times with TBS-T (Tris-buffered saline and Tween 20containing Tween 20) and detected with an image analyzer using ECL(enhanced chemiluminescence) solution.

As a result of FIG. 5, it was confirmed that the proliferation of theHSP90 client protein was inhibited in a concentration-dependent mannerby treatment with Compound 21f. The expression was significantlydecreased at 0.5 μM for Her2, Met, and Akt, and 0.1 μM for EGFR. Inaddition, expression of HSP70 was also increased at 0.5 μM.

While this invention has been described in connection with what ispresently considered to be practical exemplary embodiments, it is to beunderstood that the invention is not limited to the disclosedembodiments, but, on the contrary, is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

What is claimed is:
 1. A compound of Chemical Formula 2 or apharmaceutically acceptable salt thereof:

wherein R₁ is isopropyl, and R₂ is

R₃ is methyl, and R₉ is methyl, ethyl, or propyl.
 2. A method oftreating heat shock protein 90 (HSP90)-mediated disease in a subject inneed thereof, comprising administering an effective amount of apharmaceutical composition comprising a compound of following ChemicalFormula 2 or a pharmaceutically acceptable salt thereof, as an activeingredient:

wherein R₁ is isopropyl, and R₂ is

R₃ is methyl, and R₉ is methyl, ethyl, or propyl, to the subject,wherein the heat shock protein 90 (HSP90)-mediated disease is selectedfrom the group consisting of cancer disease, degenerative neurologicaldisease, and viral infection.
 3. The method of claim 2, the cancerdisease is any one selected from the group consisting of non-small celllung cancer, breast cancer, ovarian cancer, uterine cancer, pancreaticcancer, lung cancer, gastric cancer, liver cancer, colon cancer, skincancer, head or neck cancer, brain cancer, laryngeal cancer, prostatecancer, bladder cancer, esophageal cancer, thyroid cancer, kidneycancer, rectal cancer, acute myelogenous leukemia, chronic myelogenousleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia,and blood cancer.
 4. The method of claim 2, wherein the degenerativeneurological disease is any one selected from the group consisting ofstroke, paralysis, memory loss, memory impairment, dementia,forgetfulness, Parkinson's disease, Alzheimer's disease, Pick's disease,Creutzfeld-Kacob disease, Huntington's disease, and amyotrophic lateralsclerosis.